Packaging material and methods of using the same

ABSTRACT

The present disclosure is directed to films. The films can include polyamic acid (PAA). Methods of making and using the film for food product coverings is also included.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.15/987,198, filed on May 23, 2018, which claims benefit of U.S.Provisional Application 62/509,919, filed on May 23, 2017, the contentsof which are incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grants CBET1230189 and DMR 1007900 awarded by the National Science Foundation. Thegovernment has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as34570A_SequenceListing.txt of 33 KB, created on Sep. 29, 2020, andsubmitted to the United States Patent and Trademark office via EFS-Web,is incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

Smart packaging requires the packaging materials to provide simultaneousactive protection and intelligent communication with food and otherperishable materials. In that respect, packaging materials shouldperform the dual role of sensing and packaging.

Smart packaging requires that the packaging materials provide activeprotection and intelligent communication about the packaged food.Package materials add extra protection for the food by providinginformation about time and past conditions of the food. Intelligentpackaging advances communication capabilities of traditional packagingmaterials by providing information about the integrity and quality ofthe packaged foods and its surrounding environment from packaging, tostorage, transport and market shelves. Current smart packaging usesradio frequency identification, and indicators of environmental factorssuch as pH and heat. Even though these are commercially available, thecost is still high for large scale applications.

In contrast to intelligent packaging, active packaging does not provideinformation about the condition of packaged food, but enhances theshelf-life through a variety of mechanisms, including, but not limitedto, moisture absorption, antimicrobial packaging material, antioxidants,carbon dioxide emitters and oxygen scavengers.

Further, most food packaging materials in use are derived from startingmaterials that are either obtained from petrochemicals or they requirethe use of organic toxic solvents. The resulting polymers are notbiodegradable.

Currently, there is no practical food packaging system that integratesintelligent and active capabilities and is also biodegradable.

Therefore, what is desired is a film and film material that can be usedfor, among other uses, food packaging, that provides intelligent andactive capabilities.

Embodiments of the present disclosure provide devices and methods thataddress the above and other issues.

SUMMARY OF THE DISCLOSURE

The present disclosure is directed to films. The films can includepolyamic acid (PAA). Methods of making and using the film for foodproduct coverings is also included.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this application contains at least one drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee.

The present disclosure will be better understood by reference to thefollowing drawings of which:

FIG. 1a is an illustration of the synthesis of PAA and ternary PAAcopolymers.

FIG. 1b is an illustration of the synthesis of PAA.

FIGS. 2a-2q are illustrations of NMR data.

FIGS. 3a-3f are illustrations of NMR data.

FIG. 4 is an illustration of NMR data.

FIGS. 5a-5b are illustrations of NMR data.

FIG. 6 is a graphical illustration of diffusion coefficients.

FIG. 7 is a graphical illustration of the IR spectrum of different PAAco-polymers.

FIGS. 8a-8h are illustrations of NMR data.

FIG. 9 is illustrations of different chemical structures.

FIGS. 10a -10 aj is photographs of various PAA films.

FIGS. 11a-11l is photographs of various PAA films.

FIGS. 12m-12p is photographs of various PAA films.

FIGS. 13a-13b are graphical illustrations of absorbance andtransmittance values of various PAA films.

FIGS. 14a-14b are graphical illustrations of absorbance and emissionvalues of various PAA films.

FIGS. 15a-15e are graphical illustrations of fluorescence values ofvarious PAA films.

FIGS. 15a-15e are graphical illustrations of fluorescence values ofvarious PAA films.

FIGS. 16a-16b are graphical illustrations of fluorescence values ofvarious PAA films.

FIGS. 17a-17e are graphical illustrations of fluorescence values ofvarious PAA films.

FIGS. 18a-18d are graphical illustrations of fluorescence values ofvarious PAA films.

FIGS. 19a-19d are graphical illustrations of fluorescence values ofvarious PAA films.

FIGS. 20a-20d are graphical illustrations of fluorescence values ofvarious PAA films.

FIGS. 21a-21d are graphical illustrations of fluorescence values ofvarious PAA films.

FIGS. 22a-22d are graphical illustrations of absorbance and fluorescencevalues of various PAA films.

FIGS. 23a-23e are graphical illustrations of fluorescence values ofvarious PAA films.

FIG. 24 is a graphical illustration of absorbance values of various PAAfilms.

FIGS. 25a-25c are graphical illustrations of intensity values of variousPAA films.

FIGS. 26a-26g are graphical illustrations of intensity values of variousPAA films.

FIG. 27 is a graphical illustration of emission values of various PAAfilms.

FIGS. 28a-28c are graphical illustrations of absorbance and intensityvalues of various PAA films.

FIG. 29 is a photograph of the solubility of ternary PAA membranes inbasic solutions.

FIG. 30 is a photograph of the color changes of ternary PAA membranes inresponse to alcohol exposure.

FIG. 31 is a photograph of various PAA films.

FIG. 32 is a photograph of color changes in response to alteration inenvironmental conditions that can be further advanced with pH-dependentdyes.

FIG. 33 is a photograph of an aged PAA film.

FIG. 34 is a photograph of a four-probe and Ohm meter forcharacterization of electronics properties.

FIGS. 35a-35c is a photograph of PAA films.

FIGS. 36a-36b are graphical illustrations of voltage vs. current ofvarious PAA films.

FIGS. 37a and 37b are SEM images of PAA films.

FIGS. 38a-38h are graphical illustrations of voltage vs. current ofvarious PAA films.

FIG. 39 is a digital image of the oil-permeability test.

FIG. 40 is a digital image of the water-vapor permeability test.

FIGS. 41a-4j are illustrations of NMR data.

FIG. 42 is a picture of a macroscopic and four microscopic pictures ofTrichaptum biforme.

FIG. 43 is microscopic pictures of Fusarium oxysporum.

FIGS. 44a-44b are graphical illustrations of membrane loading in a welland cytotoxicity.

FIGS. 45a -45 av are photographs of various PAA films.

FIGS. 46a-46b are SEM images of various PAA films.

FIGS. 47a-47h are photographs of various PAA films.

FIGS. 48a-48b are SEM images of various PAA films.

FIGS. 49a-49b are SEM images of various PAA films.

FIGS. 50a-50b are SEM images of various PAA films.

FIGS. 51a-51b are SEM images of various PAA films.

FIGS. 52a-52b are SEM images of various PAA films.

FIGS. 53a-53b are SEM images of various PAA films.

FIGS. 54a-54d are SEM images of various PAA films.

FIGS. 55a-55f are SEM images of various PAA films.

FIGS. 56a-56b are SEM images of various PAA films.

FIGS. 57a-57b are SEM images of various PAA films.

FIGS. 58a-58m are illustrations of different chemical structures.

FIG. 59 is a graphical illustration of phase-inversion incoagulation-bath.

FIGS. 60a-60p are photographs of various PAA films.

FIGS. 61a-61e are photographs of various PAA films.

FIGS. 62a-62g are photographs of various PAA films.

FIGS. 63a-63k are photographs of various PAA films.

FIGS. 64a-64d are SEM images of various PAA films.

FIGS. 65a-65d are photographs of various PAA films.

FIG. 66 is a table of various PAA films' properties.

FIGS. 67a-67e are photographs of various PAA films covering variousfoods.

FIGS. 68a-68c are photographs of various PAA films covering variousfoods.

FIG. 69 is a graphical illustration of a color change of a PAA film.

DETAILED DESCRIPTION OF THE DISCLOSURE

In the discussion and claims herein, the term “about” indicates that thevalue listed may be somewhat altered, as long as the alteration does notresult in nonconformance of the process or structure to the illustratedembodiment. For example, for some elements the term “about” can refer toa variation of ±0.1%, for other elements, the term “about” can refer toa variation of ±1% or ±10%, or any point therein.

As used herein, the term “substantially”, or “substantial”, is equallyapplicable when used in a negative connotation to refer to the completeor near complete lack of an action, characteristic, property, state,structure, item, or result. For example, a surface that is“substantially” flat would either completely flat, or so nearly flatthat the effect would be the same as if it were completely flat.

As used herein terms such as “a”, “an” and “the” are not intended torefer to only a singular entity, but include the general class of whicha specific example may be used for illustration.

As used herein, terms defined in the singular are intended to includethose terms defined in the plural and vice versa.

Reference herein to any numerical range expressly includes eachnumerical value (including fractional numbers and whole numbers)encompassed by that range. To illustrate, reference herein to a range of“at least 50” or “at least about 50” includes whole numbers of 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers 50.1,50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In a furtherillustration, reference herein to a range of “less than 50” or “lessthan about 50” includes whole numbers 49, 48, 47, 46, 45, 44, 43, 42,41, 40, etc., and fractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4,49.3, 49.2, 49.1, 49.0, etc. In yet another illustration, referenceherein to a range of from “5 to 10” includes whole numbers of 5, 6, 7,8, 9, and 10, and fractional numbers 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, etc.

As used herein, the term “film” refers to a thermoplastic film madeusing a film extrusion and/or foaming process, such as a cast film orblown film extrusion process. For the purposes of the present invention,the term includes nonporous films as well as microporous films. Filmsmay be vapor permeable or vapor impermeable, and function as liquidbarriers under normal use conditions.

As used herein, the term “thermoplastic” refers to polymers of athermally sensitive material, which flow under the application of heatand/or pressure.

As used herein, the term “polymers” includes, but is not limited to,homopolymers, copolymers, such as for example, block, graft, random andalternating copolymers, terpolymers, etc. and blends and modificationsthereof. Furthermore, unless otherwise specifically limited, the term“polymer” shall include all possible geometrical configurations of thematerial. These configurations include, but are not limited toisotactic, syndiotactic and atactic symmetries.

Biodegradable Ternary Co-polymers of Conducting Electroactive PAAMembranes hereby referred to as membranes or films. For descriptivepurposes, the term membrane has the same definition as that of the term“film” discussed above.

Polyamic acid (PAA) is a polymer that has many novel properties. PAA iselectroactive, substantially biodegradable and has free carboxyl andamide groups that can act as molecular anchors. PAA can also be used inconjunction with both organic and inorganic solvents due to itssubstantial chemical resistance. PAA is a generic name of use for thepolycondensation product of dianilines and dianhydrides synthesized inanhydrous organic aprotic polar solvents.

The present disclosure is directed to PAA films and PAA films as foodpackaging materials that can provide both active-packaging qualities andintelligent-packaging qualities. These PAA films and PAA films as foodpackaging materials can be formed without any petroleum based orpetrochemical ingredient and/or any ingredient formed from a hydrocarbonand/or without an organic solvent.

The PAA films were created from compositions including biologicalcompounds (e.g. amino acids, sugars) and one or more of intrinsicantimicrobial agents (e.g. sulfanilic acid, p-aminosalicylic acid), andcross-linkers (e.g. glutaraldehyde, carbodiimidazole) in the presence ofother substances, for example diamines and dianhydrides. Also, PAA canbe further modified into polyimide depending on the processingconditions or employed as stabilizing agents during nanoparticlessynthesis.

H DOSY NMR studies showed that the average molecular weight of PAA filmswere between about 10⁶ and about 107 Da while average molecular weightof regular PAA polymer was about 1.43×10⁵ Da.

PAA has advanced mechanical properties in the range of about 2.2-about2.7 GPa modulus elasticity comparable to strong plastics (2.4 to 3.2GPa).

PAA also demonstrates stability in common solvents, high opticaltransparency, impermeability to gas exchange, oil and water vaportransfer.

FIG. 66 is a table of modulus elasticity, tensile strength andelongation of six films of the present disclosure (bottom six films onthe list) as compared to other non-PAA films.

Non-crude oil-based plastic PAA films illustrate voltage changes inresponse to pH change. Showing a trend in response to pH changedemonstrates the intelligent properties of the packaging material of thepresent disclosure, which does not require complicated sensorelectronics to indicate food freshness/quality. In the table below, itcan be also seen that there is a voltage change in the disclosed filmsas a function of their thickness.

TABLE A Membrane Type Thickness [mm] DC Voltage [mV] PAA-A-GA 0.02 −0.7PAA-W-GA 0.02 1.2 PAA-BB-GA 0.02/0.06 −0.4/0.4   PAA-PCI-GA 0.06 0.9PAA-C-GA 0.02 1.8 PAA-DA-GA 0.03 0.1 PAA-PCI-GA 0.06/0.12 −0.6/−1.1PAA-W-GA 0.02 2.4 PAA-A-GA 0.05 0.4 PAA-DP-GA 0.05 −0.4 PAA-pAB-W-GA0.06 −0.9 PAA-pAB-GA 0.06 −0.5/−0.4 PAA-A-GA 0.09 −0.6

In Table A PAA: Poly (amic) acid; GA: Glutaraldehyde and the letters inthe middle refer to different small molecules such as A-alanine andW-tryptophane. As referred to herein, the term small molecule can referto any organic molecule having a low molecular weight of less than about900 Daltons) that may regulate a biological process, with a size on theorder of about 1 nm. Type of small molecule and concentration ofglutaraldehyde affect voltage of dry PAA membrane's potential, and itsbehavior against changes as a function of pH and salt concentrations.

Microbiological tests showed that there was no bacterial developmentwhich means that PAA copolymer films developed in connection with thepresent disclosure worked as a strong active packaging material. As apackaging material, the PAA films can be provided on a roll, the filmprovided with a predetermined width and a predetermined length. Anexample of an existing roll of this type would be a roll of Saran™ Wrap,having a width of about 12″ and a length of tens or hundreds of feet.Rolls of the disclosed PAA films can be wider or narrower, and can alsobe longer or shorter than this example, as desired.

The disclosed PAA films can be applied so as to cover an entire foodproduct, or a portion thereof. The PAA films can also be provided so asto contact the entire food product or a portion of the food product, orso as to not contact any portion of the food product due to anintervening material or a space between the food product and the PAAfilm. The disclosed PAA films can be applied by a user and/or thedisclosed PAA films can be applied by a packaging device/machine.

As discussed below, packaged food products did not include color changesor fungal development, this is related to the non-porous nature of PAAfilm, which did not allow air and water vapor entry. Measured voltage(0.2 mV) did not show any changes which means that there was no foodspoilage and decomposition during the tested times.

The utilization of organic solvents such as ethanol is generally notpreferred in the synthesis of PAA since they are nucleophiles and cancompete with the dianiline component to attack the dianhydrideresources. In the present disclosure, the use of ethanol and even wateras part of the solvent system did not show any effect on the formationof the PAA polymer when solid dianhydride was added to an alreadydissolved dianiline. This represents a major deviation from standardchemistry of PAAs and one that has led to the preparation of a new classof stable polymeric compositions and novel processing procedures asreported here. We however, observed the (FIGS. 2a-2q ) formation ofester and alteration in the repeating units of PAA. As shown in Table B,the utilization of ethanol as part of the solvent system significantlyimproved the mechanical properties of the synthesized films/membranes.

TABLE B Selection of solvent and formation of viscous PAA solution.Mixture of solvent Observation 50:50 or 35:65

High viscosity, require warning (i.e. 50° C.), DMAC:EtOH resulting inmembranes that are strong but limited colors (no blue color obtained)25:75

Medium viscosity, require warning DMAC:EtOH (i.e. 60° C.), resulting inmembranes that are strong, but limited colors 35:50:15 or Mediumviscosity, require warning 30:50:20

(i.e. 60° C.), produces membranes with broad DMAC:EtOH:Water range ofcolors. The color intensity is stronger than those prepared using DMAC.60:40

Did not form PAA viscous solution but DMAC:Water resulted in yellowprecipitate. This did not result in any membrane formation. 60:30:10

Did not form PAA viscous solution but DMAC:Water:AcOH resulted in yellowprecipitate. This did not result in any membrane formation. Acetic acidlimits the role of GA.

indicates data missing or illegible when filed

It should be noted that heating was not continuous; rather it wasstopped right after PMDA was added to the dissolved 4,4′-oxydianiline(ODA). Continuation of heating resulted in highly viscous PAA solutionwhich does not allow membrane formation.

Another important observation noted here was that the average molecularsize of PAA polymers decreased when the solvent system changed from DMACto DMAC/Ethanol, and further decreases were observed forDMAC/Ethanol/Water system.

Parameters relating to the synthesis of FIG. 1 a membranes wereevaluated at four aspects(1)-(4).

(1) Formation of Amorphous, Glassy and Plasticized Membranes

When pure PAA viscous solution (either from ODA+PMDA or PDA+PMDA) wascasted on glass to form membrane, the fate of the membrane was shown tobe determined by evaporation mediated solvent elimination andsolvent-nonsolvent exchange in coagulation bath.

TABLE C Effect of evaporation period on PAA membrane preparationIncubation time (h)¹ Texture Character <4 h Amorphous Similar to phaseinverted PAA membrane in coagulation-bath 4-8 h Glassy The outer surfaceis shiny, but not totally plasticized. The membrane turns into brittleform within 2 h right after being taken out from the coagulation-bath6-10 h Mix Mostly the outer layer is fully plasticized while the innerpart is amorphous. The membranes are durable, and never turn into aglassy form >12 h Plasticized Plastic-like transparent membrane, durableand flexible. Coagulation-bath doesn't affect appearance of the membraneIncubation time refers to the time-period when membranes were incubatedunder a hood at 80 rpm/min face-shield. In all cases, the coagulationbath employed was pure-water. ¹Thickness and viscosity of the casted PAAsolution affected the time requirement, but 12 h or over were enough toobtain plasticized membranes for the casted solutions at up to 2 mm(beyond this point, thicker membranes were not tested) thickness. Forthin membranes (e.g. below 50 μm), 6 h was enough to obtain fullyplasticized membranes. Evaporation of the solvent is the main elementdetermining the fate of the membrane's texture. This is furtherdiscussed below. However, the most prominent parameter is the humidityof the surrounding environment. However, pre-heating the casted PAAsolution decreases the negative effect of the humidity, which can leadto accelerated removal of DMAC coupled with enhanced GA activity.

(2) Crosslinker Effect on Membrane Formation

In accordance with the present disclosure, glutaraldehyde (GA) was usedas the cross-linker due to the fact that GA provided the most pronouncedeffect on PAA membrane formation.

TABLE D Effect of evaporation period on PAA-GA membrane preparationIncubation time (h)¹ Texture Character <2 h Amorphous Similar to phaseinverted PAA membrane in coagulation-bath 4-6 h Glassy The outer surfaceis shiny, but not totally plasticized while the edges are totallyplastic-like. The membrane turns into brittle form within 1 h rightafter taken out from the coagulation-bath. The brittle form shows veryhigh glassy character. 4-8 h Mix The outer layer is fully plasticizedwhile the inner part is amorphous. Only thicker membranes (e.g. over 2mm) forms this type of membranes. These ultimately turns into glassy-brittle form within days. >8 h Plasticized Plastic-like transparentmembrane, durable, flexible and non-soluble in common organic solvents.Coagulation-bath does not affect appearance of the membrane ¹Viscosityof the casted solution is determined by GA activity. Other than GA,other crosslinkers were also used as detailed below.

The time difference between transformations from amorphous to glassytexture were linked to the degree of crosslinking. This is attributed tothe fact that the cross-linker is becoming an element in determining thefate of the membrane in terms of color and texture. It is not criticalthat the membrane loses a higher proportion of the DMAC in order to formthe plasticized PAA membranes. This is related to cross-linking ofindividual PAA membranes with crosslinker (i.e. glutaraldehyde). Forexample, in the case of p-phenylenediamine (PDA)-PMDA based PAAmembrane, 30 min incubation is sufficient to provide the plasticized PAAmembranes unlike the ODA-PMDA based PAA membrane that requires over 4 hincubation. This is expected because PDA has two amino groups whichenhance its cross-linking with GA. Even though, no chemical treatmentwas performed in DMAC, the final forms of the membranes even forrelatively lower GA concentrations (pre-diluted in DMAC) were stillobtained in the plasticized form. This was not common for the GAconcentrations that were directly added from stock. For example, in thecase of PAA-CS-GA membrane, the same amount of GA when dissolved inwater produced an amorphous membrane while GA that was pre-diluted inDMAC provided plasticized membrane. Further, heat treatment to GA/PAAmembranes resulted in plasticized membranes, which can be related to thepromotion of cross-linking and faster evaporation of solvent. Furtherdetails are provided below.

(3) Small-Molecule Effect

None of the small molecules showed any strong impact on membraneformation when added to the casted PAA solution without the co-additionof the cross-linker. Increase in viscosity related to the addition ofsmall molecule (excluding the cross-linker GA) did not affect theoverall membrane formation (amorphous, glassy or plasticized) asdetailed below. However, the use of small molecules in the presence ofthe cross-linkers significantly impacts the structure, the plasticityand other notable physical attributes of the resulting membranes.

As shown below, while certain small molecules with PAA copolymers endedup as plasticized membranes, others were amorphous in nature. Similarly,the mechanical properties of the membrane under same conditions showeddirect dependence on type of the small molecule employed.

This above chart illustrates the effect of small molecule on membraneformation. (i) Selection of small molecule is not limited to these smallmolecules; (ii) GA concentration has strong influence on the final formof the membrane apart from the small molecule used in the study.

The addition of glutaraldehyde is an element in the kinetics of membraneformation. For example, at 0.5% GA concentration, the time needed toform a stand-alone membrane diminishes. This change is believed to becoming from the alteration in the characteristics of the solutionitself. For example, PAA alone requires 12h to form a membrane of PAAalone; PAA-GA requires 8h while PAA-GA-SA requires 4h to givestand-alone (FIG. 1a ) membranes. The time requirement for providingstand-alone membrane is subject to change in response to thickness ofthe casted solution. However, it should be noted that obtainingstand-alone membrane in shorter period may not be related to fasterdrying.

When the stand-alone membrane is first obtained, its mechanical propertyis poorer than the membranes that are fully dried. The modulus ofelasticity and tensile strength are the main parameters that improveddramatically when the membrane is fully dried. In contrast to this, %elongation decreases at least two-times upon total drying, which wasobserved for PAA-I-GA, PAA-K-GA, PAA-CA-GA or thicker PAA-GA (over 2 mmcasted solution) membranes; when they get dry, they show very highglassy character which makes them as brittle as glass.

L-alanine and L-cysteine in all cases of provided plasticized PAAmembranes, and L-tryptophan-methyl ester also providedplasticized-membranes if the conditions are controlled in terms ofhumidity and heat. However, utilizing higher concentrations of GA (i.e.2% or higher) for any type of small molecule co-polymerized with PAAresulted in plasticized PAA membranes. These also affect the formationof colorful membranes. Here, for example, L-alanine gives green membranewhile PAA-GA gives chestnut color membrane. Actually, PAA-A-GA providedthe membranes which were the best examples of plasticized membranes forFIG. 1a membranes, which was also comparable to the membranes obtainedfrom FIG. 1b . Regarding color formation, even for PAA-small moleculeco-polymers, the age of GA can influence the results.

(4) Final Step of Coagulation Bath

TABLE E Effect of coagulation bath on membrane surface characteristics.Coagulation-bath Surface-characteristics Pure-water Shiny, porous ornon-porous Methanol^(1,2) Sponge, nano-fabric or porous Ethanol^(1,3)Sponge, nano-fabric or porous Ethanol-water mixture¹ Sponge andnon-porous In Table E, ¹For extended evaporation times, sonication mightbe required in order to obtain nano-fabric and/or sponge surfaces. ²Themembranes, which are giving glassy texture/form in the case ofpure-water coagulation bath, give sponge or non-porous surfaces anddurable membranes. ³In the case of pure-water coagulation bath, themembranes becomes brittle within times.

Membranes possessing nano-porous, sponge, nano-fabric, non-porous andfeatureless surfaces can be obtained using the method depicted in FIG.1a , which are detailed below.

Characteristic differences exist between the methods depicted in FIG. 1band FIG. 1a . These differences are discussed below and throughout theapplication. These include (i) evaporation is the main driving force forphase-inversion, (ii) small-molecules, which are co-polymerized with PAAare dissolved in PAA viscous solution or pre-dissolved in a solventprior to being co-polymerized with PAA, (iii) small-molecule can becross-linked with the cross-linker in order to adapt the overallproperties of the resulting membrane, and (iv) flexible design viacombination of small molecules and/or the order of the addition of thesmall molecule or cross-linker.

GA is a cross-linker. Since GA can exist in different chemical forms inaqueous and organic solvents (as detailed below) it is adapted for theobjectives met by the present disclosure. For example, GA can bepolymerized into a water-soluble and non-soluble forms based on theobjective. Here, GA was first aged through incubating the solution at70° C. for hours. Optimization was followed with ¹H NMRcharacterization. As detailed below, the following manipulations wereperformed for GA to obtain the desired membranes;

-   -   i. Utilization of aged or non-aged GA. While aged GA is used in        order to obtain fluorescently active membranes, non-aged GA was        preferential to obtaining physically strong membranes.    -   ii. Quenching GA activity with methanol or ethanol is needed in        order to obtain physically strong membranes. The addition of        methanol results in physically strong and non-soluble membranes.    -   iii. Concentration of GA or heat-treatment of GA before it is        introduced to PAA solution has strong effect on membrane        formation with respect to color and time-requirement for        stand-alone membrane formation.    -   iv. Very high concentrations of GA (i.e. over 2%) prevent the        formation of ideal long-lasting membranes; higher GA makes PAA        membranes brittle and even in some cases disrupts proper        membrane formation. In the case of imidazole, concentrations can        vary as described below.

As used herein, the term “fresh” or “non-aged” GA refers to GA purchasedfrom companies, which were used as received and stored at all times atabout −20° C. The term “aged” GA refers to GA that was kept in an ovenfor about 1-2 hours (e.g. 50-70° C.) prior to use. The term “over-aged”refers to GA that was stored at room temperature for about 2 weeks orlonger.

The data and discussion below presents the development, processing,characterization and novel applications of the disclosed films. Due tothe organic solvents being environmental pollutants, replacing them withsubstantially environmentally benign solvents are desired.

1D and 2D NMR techniques indicated that DMAC/EtOH and DMAC/EtOH/Watersolvent mixtures were applicable for generating PAA polymers synthesizedin DMAC. Reducing the use of DMAC by about 75% did not affect the PAAsynthesis. However, the repeating units were altered as cis-/trans-ratioand the average molecular weights of the PAA polymers decreased by up to5 times. The use of crosslinkers, especially GA, was utilized to alterthe kinetics of the phase-inversion. GA is a component in the synthesisleading to the formation of amorphous and plasticized membranes. Smallmolecules were co-polymerized with PAA to manipulate the overallproperties of the membrane with respect to their plasticity,antimicrobial properties and mechanical strengths, as discussed indetail below.

The methods, apparatus and compositions of the present disclosure willbe better understood by reference to the following Examples, which areprovided as exemplary of the disclosure and not by way of limitation.

Example 1.1-Materials and Methods

All of the reagents used in this and the following examples werepurchased from Sigma-Aldrich (St. Louis, Mo.). Escherichia coli ATCC®25922 Citrobacter freundii, ATCC® 8090 and Staphylococcus epidermidisATCC® 12228™ were purchased from American Type Culture Collection (ATCC)(Manassas, Va., USA). Dimethyl sulfoxide (DMSO)-d₆, was purchased fromCambridge Isotope Laboratories (Andover, Mass. USA). Unless otherwisespecified, phosphate saline (PBS) buffer was used as 50 mM pH 7.2. Allsolutions were prepared with triply distilled Nanopure water withresistivity of 18 MΩ.

The above figure illustrates the steps in the synthesis of a PAA Polymerof the present disclosure using optional solvent systems: The ratio of4,4′-oxydianiline (ODA):PMDA was tested from 1.20:1.00 to 1.00:1.05. Theratio given for each solvent was used through the examples.

The synthesis of PAA films in accordance with the present disclosure isshown in FIG. 1a . In the process of FIG. a, crosslinkers serve as areactive transforming agent that crosslinks re-organize the kinetics ofthe membrane formation and define the fate of the membrane.

The superscript numbers herein refer to the steps of the correspondingsuperscript numbers in FIG. 1a . In FIG. 1a , the preparation of PAA andTernary PAA co-polymers is illustrated. MeOH refers to methanol and DMACrefers to N,N′-dimethylacetamide. ¹Examples include D-glucosamine,L-lysine, L-alanine and other amino acids. ²Glutaraldehyde stock wasobtained in water, but throughout the study it was added aspre-dissolved in methanol or DMAC to possibly alter its activity bychanging the working microenvironment. ³Since pre-treating GA with MeOHor DMAC affected the activity; their incubation at room temperature wastaken at periodic intervals. But incubation time was changed just toalter the resulting surface properties of the PAA membrane.⁴Phase-inversion in ethanol/water mixture was applied to alter thesurface properties of the resulting membrane. ⁵The last step ofphase-inversion took place in nano-pure water, followed by drying underhood.

Further synthesis of PAA films is shown in FIG. 1b . The superscriptnumbers herein refer to the steps of the corresponding superscriptnumbers in FIG. 1 b. ¹Methanol can be added to the system at this stage;²This procedure was only used for amino acids and glucosamine; ³Methanolcan be added to the system immediately after the introduction ofGA-biomolecule; ⁴The membrane can be sonicated inmethanol/ethanol/methanol-water mixture. ⁵The small molecules,4-amino-2-chlorobenzoic acid, p-aminobenzoic acid and aminosalycilicacid, could be added to the system during PAA synthesis. Films generatedusing the synthetic method of FIG. 1b were used for food-packagingthroughout the examples.

Example 1.2-Structural Characteristics of PAA Films

The PAA film and the functionalized derivatives were dissolved in DMSOd6(unless otherwise stated) and then subjected to 1H Nuclear MagneticResonance (NMR), 13C NMR, and 1H-correlation spectroscopy (COSY), 1H 13CHeteronuclear Single Quantum Coherence (HSQC), 1H 15N HSQC, 1H 13CHeteronuclear Multiple Bond Coherence (HMBC) and 1H 15N HMBCcharacterizations. A Bruker AM 600 spectrometer operated by Topspin™ 3.0NMR software was used for spectra measurement and analysis.

In order to fully annotate structure of phase inverted PAA and thedesigned PAA, NMR and IR experiments were performed. NMR was also usedto monitor the possibility of Bisphenol A formation in relation to heattreatment and exposure time.

To move step by step, ACD/ChemSketch (Freeware) academic edition wasused to draw the PAA structures, and PAA-GA interaction. This was due tothe fact that in all cases, GA was used as an element in preparation ofPAA films.

In the above structures, the proposed structure of PAA polymers areshown. [A]_(a)-[B]_(b)-[A]c-[B]_(d) [a, b, c and d can be 1 or more, andcan be the same or different]. In the case of PAA synthesized in DMAC,[A]₂-[B]₃-[A]₂-[B]₃ is proposed as the possible structure.

TABLE F ¹H NMR of PAA and ternary PAA Films Carboxyl Amino CarbonylAromatic Aliphatic Film group group group protons proton PAA 13.0510.56/10.53 N/A 8.35/8.00/7.74 N/A 7.72/7.05 PAA¹ Not 10.57/10.54 N/A8.37/8.02/7.77 N/A visible 7.73/7.06 PAA-GA 13.33 10.55/10.52 9.268.34/7.99/7.74 Not clear 7.71/7.05 PAA-GA² 13.54 10.65/10.549.30/9.22/9.1/8.75 8.33/7.97 5.00/4.92/4.87 10.52 7.70/7.044.68/1.77/1.23 PAA-GA- Not 10.66/10.55 9.32/9.28 8.35/8.00 4.68/3.51SA^(1,2) visible 10.53 7.72/7.05 1.66/1.22 PAA-GA- 13.00 10.65/10.549.32/9.30 8.33/7.97/7.76 5.26/2.03/1.23 SA-pAS² 10.51 7.70/7.040.93/0.83 PAA-GA- 13.19 10.65/10.53 9.32/9.31/9.29 8.33/7.98/7.722.03/1.91/1.90 SA-pAS-A 10.51 9.06/9.04 7.70/7.04 1.39/1.23 PAA-GA-13.19 10.65/10.53 9.39/8.74 8.33/7.97 5.74/2.08/1.23 SA-pAS^(2,3) 10.517.70/7.04 PAA-GA- 13.16 10.66/10.54 9.29/9.22 8.33/7.97/7.726.09/5.97/4.32 SA⁴ 10.51 7.70/7.04 3.69/3.45/1.32 1.23/1.05

TABLE G ¹³C NMR of PAA and ternary PAA film Film Carbonyl Carboxl Amidearomatic Aliphatic PAA 165.79/ 166.73/ 152.95 141.20/139.08/134.81 N/A165.69 166.42 133.12/129.83/128.86 127.56/121.53/118.83 PAA¹ 165.89/166.81/ 152.05 141.29/139.18/134.87 N/A 165.79 166.51133.99/133.18/130.90 129.1/128.95/127.64 121.63/120.33/118.91117.61/116.58 PAA- 165.77/ 166.68/ 152.94 141.21/139.08/134.79 Not seenGA 165.66 166.38 133.06/129.05/128.83 127.53/121.51/118.81 118.29 PAA-165.70/ 166.62/ 152.86 141.14/139.01/134.74 Not seen GA² 165.59 166.31133.71/129.02/128.77 167.59/ 127.49/121.44/121.35 167.30 118.74/118.72PAA- 165.86/ 166.75/ 153.02 141.28/140.68/139.16 37.57 GA- 165.75 166.46136.52/134.84/133.18 SA^(1, 2) 167.28/ 166.36 130.83/129.13/128.91167.89 121.60/118.88 PAA- 165.72/ 166.63/ 152.89 141.17/140.45/139.04Not seen GA- 165.61 166.33 134.75/133.02/130.72 SA- 129.03/128.78/127.49pAS-A 121.47/120.06/118.76 118.74 PAA- 165.73/ 166.63/ 152.89141.18/140.47/139.05 37.47 GA- 165.63 166.33 134.75/131.66/130.72 SA-167.60/ 129.04/127.49/121.48 pAS^(2, 3) 167.27 120.05/118.77/118.74 PAA-165.72/ 166.65/ 152.88 141.16/139.03/134.76 13.78 GA- 165.62 166.34133.05/130.75/129.02 SA⁴ 128.79/127.50/121.62 121.46/118.76/118.74 Thefollowing superscript numbers refer to the tables above: ¹very highconcentration (150 mg/mL) of PAA; ²GA used high concentration 2%. ³highconcentration (80-100 mg/mL) of pAS. ⁴PAA was synthesized in 65:35,Ethanol:DMAC. Protons of N,N′-dimethylacetamide were not listed on thetable since they are only impurities.

Tables F and G provide a comparison for PAA alone vs. various PAA films.Since GA and small molecules were used at very low amount in comparisonto PAA, ¹H and ¹³C NMR techniques did not provide the presence of newpeaks for each group. However, at higher amount of sulfanilic acid andglutaraldehyde, the characteristic peaks related to these were observed.

NMR data is shown in FIGS. 2a-2q . Specifically FIG. 2a is NMR data for¹H, FIG. 2b is NMR data for ¹H COSY, FIG. 2c is NMR data for ¹H-¹³CHSQC, FIG. 2d is NMR data for ¹³C, FIG. 2e is NMR data for ¹H-¹³C HMBS,FIG. 2f is NMR data for ¹H-¹⁵N HSQC, FIG. 2g is NMR data for ¹H-¹⁵N HMBCand FIG. 2h is NMR data for co-presentation of ¹H-¹³C HMBC and ¹H-¹³CHSQC spectra (blue HMBC and red HSQC). Further NMR data for PAA membranephase-inverted in pure-water in FIG. 2i , which is NMR data for ¹H, FIG.2j is NMR data for ¹H COSY, FIG. 2k is NMR data for ¹H-¹³C HSQC, FIG. 2lis NMR data for ¹³C, FIG. 2m is NMR data for ¹H-¹³C HMBS, FIG. 2n is NMRdata for ¹H-¹⁵N HSQC and FIG. 20 is NMR data for ¹H-¹⁵N HMBC of PAA-GAmembrane prepared according to the method of FIG. 1 b.

In this disclosure, NMR data is used to obtain physical, chemical,electronic and structural information of the disclosed of organiccompounds. It is due mostly to the chemical shift on the resonantfrequencies of the nuclei present in the compound compared to areference magnetic field (usually tetramethylsilane or TMS). Chemicalshift is the function of the nucleus and its environment, which ismeasured relative to a reference compound (i.e. TMS). As for thespecific NMR data presented in the figures of this disclosure, PAA doesnot give peaks at the following region including aliphatic region(single or double bond). So, any missing PAA signature peak is anindication that the polymer is not present or is degrading. The NMRimages also provide how GA binds to the PAA molecules.

The disclosed data is used to provide detailed information on thetopology, dynamics and three-dimensional structure of molecules. The NMRdata in FIGS. 2a-2o compare the NMR spectra of PAA and PAA-GA. Thefigures generally illustrate the chemical interaction of GA with PAAbeing due to cross linking between GA and PAA.

¹H NMR spectrum (FIG. 2a ) of PAA depicts presence of carboxyl, aminoand aromatic protons. The aliphatic protons are from residualN,N′-dimethylacetamide (DMAC). According to the depicted ¹H spectrum,only one type of carboxyl group is present in PAA polymer while twocarboxyl carbons present in PAA were revealed by ¹³C spectrum (FIG. 2b). Since the microenvironment of protons in the carboxyl group is moreisolated, it was observed as single carboxyl group.

However, an amino group proton was obtained as an overlap of two peaks(FIG. 2a ); according to ¹H-¹⁵N HSQC (FIG. 2f ) there is only one typeof nitrogen, but the nitrogen locates in two slightly distinctenvironments which explains the presence of the overlapped peak. Thiswas further supported by ¹H-¹⁵N HMBC (FIG. 2g ) spectrum wherelong-range couplings of the two overlapped amino protons showed the samelong-range couplings. The overlapped peaks at 7.75 ad 7.72 ppm were fromtwo different carbon atoms which was supported by ¹H-¹³C HSQC (FIG. 2c )and ¹H-¹³C HMBC (FIG. 2g ); the peak 7.75 ppm gave cross-peak withcarbon peak at 128.92 ppm while the peak at 7.72 ppm gave cross-peakwith carbon peak at 121.57 ppm. According to ¹H-¹³C HMBC (FIG. 2g )spectrum, both the protons gave peak at 7.75 ppm and 7.7.72 ppm showedtwo distinct long range couplings, which could not be obtained by justone proton on proton. Further, three long-range couplings were observedfor amide carbon, which were linked to the protons on pyromelliticdianhydride (PMDA) group. Therefore, all these results indicate thatseveral PAA structures can be produced in accordance with the presentdisclosure.

Glutaraldehyde (GA) can bind at different positions to PAA. According to¹H (FIG. 2i ) and ¹³C (FIG. 2j ), inclusion of GA did not affect PAAstructure, rather added new groups; particularly, the presence ofcarbonyl proton and aliphatic protons around 5 ppm and 1-2 ppm revealedthat GA chemically bound to PAA. Further indications that GA waschemically bound to PAA was obtained from ¹H COSY, ¹H-¹³C HSQC,¹H-¹³C-HMBC and ¹H-¹⁵N HMBC NMR spectra. 1H COSY spectrum gave new peaksrelated to the presence of GA. For pure PAA, there is no long rangecoupling for the amino groups with the shift at ˜8 ppm while it isstrong for GA modified PAA. Similarly, new and strong long-rangecouplings were observed for carbonyl proton and the free protons onPMDA, particularly which locates between two free carboxyl groups.¹H-¹³C HSQC revealed that GA interaction nearly eliminated the presenceof adjacent peaks nearby the peak at ˜7 ppm, which could be related tothat GA attacked on the phenyl ring of 4,4′-oxydianiline (ODA).

Similarly, the long-range couplings for the adjacent peaks got lost viaGA interaction. According to ¹H-¹⁵N HMBC NMR spectral data, nearly allof the long range couplings were lost between the amide nitrogen and theprotons on phenyl ring of ODA; particularly proximal to the amino group.However, at the same time, one of the amino peaks seen in the ¹H-¹⁵NHMBC NMR spectrum was lost; this peak stayed the same for low GAconcentrations. A new amino peak was observed at ˜10.65 ppm (FIG. 2i ).Therefore, for low levels of GA, GA prefers to attach on phenyl rings ofPAA while at high levels, GA attaches on amino groups in addition tophenyl rings.

GA preferentially binds to phenyl ring of PAA. In particular, it bindsto the ODA ring of PAA polymer. FIG. 2q illustrates that GA interactioneliminated the presence of small side peaks at aromatic region, whichbelongs to the proton of ODA. This is because PMDA has more sterichindrance and hence, the GA preferentially binds onto the ODA portion ofthe PAA molecule.

GA preferentially binds to phenyl ring of PAA. In particular, it bindsto the ODA ring of PAA polymer. This is partially illustrated in FIG. 2p, which illustrates aromatic peaks of PAA being at 7.07 ppm, 7.74 ppm,8.01 ppm and 8.36 ppm. Amino peaks of PAA are at 10.54 ppm and 10.57ppm. Carboxyl peak of PAA is at 13.51 ppm. ODA residue is shown in leftcircle and PMDA is shown in the right circle.

FIG. 2q illustrates that GA interaction eliminated the presence of smallside peaks at aromatic region, which belongs to the proton of ODA. Thisis because PMDA has more steric hindrance and hence, the GApreferentially binds onto the ODA portion of the PAA molecule. In FIG.2q , the used GA concentration was less than 5% of the PAA concentrationwhen PAA-GA membrane was prepared.

NMR data is shown in FIGS. 3a-3f Specifically FIG. 3a is NMR data for¹H, FIG. 3b is NMR data for ¹H COSY, FIG. 3c is NMR data for ¹H-¹³CHSQC, FIG. 3d is NMR data for ¹³C, FIG. 3e is NMR data for ¹H-¹³C HMBS,FIG. 3f is NMR data for ¹H-¹⁵N HSQC spectra of a PAA-SA-GA membranesynthesized according to the method shown in FIG. 1 b.

Introduction of sulfanilic acid (SA) to PAA did not produce anyadditional peaks. However, some of the interactions observed in the ¹HCOSY spectrum of PAA-GA were not observed for PAA-SA-GA. For ¹H-¹³CHSQC, one additional minor peak was observed at 8.21-130.18 ppm inaddition to PAA-SA-GA. Similarly, ¹H-¹³C HMBC gave additional minorextra interactions for the protons at 8.16 and 7.82 ppm, which were moreof long-range couplings shifted to more down-field, but simultaneouslywere protected. However, the cross-peak at 7.82-167.9 ppm could bespeculated that it was from SA, rather GA. ¹H-¹⁵N HSQC spectrum did notshow any differences. Overall, it can be said that, sulfanilic acidpeaks were not clear in the membrane, while minor differences wereobserved in 2D NMR spectra.

As can be seen in FIG. 4 PAA-GA was incubated under sun-light for over 3years in an airtight glass-container. Then, the film was dissolved inDMSO. 1H-13C HSQC spectrum clearly shows that the PAA-GA membrane lostits structural integrity, but no Bisphenol A (BPA) formation wasobserved.

BPA is primarily used to make plastics such as water bottles. There arestudies showing that BPA might mimic natural receptors in the body andthereby cause an irreversible change at the genetic levels. Based onthis potential effect, BPA and a host of other compounds were classifiedas endocrine disrupting chemicals. Certain plastics may not have BPA atthe outset but with time, they may produce BPA after extensive usage andbreakdown. The disclosed films did not produce BPA during study of theirdegradation and are therefore considered substantially safe for humanhealth and the environment.

Further, Cabot sharp cheddar cheese was wrapped in a PAA-pAS-SA-GAmembrane of the present disclosure for three months. Subsequently, wecompared the proton ¹H NMR of freshly purchased cheese (FIG. 5a ) andthe cheese kept in the membrane (FIG. 5b ). There was no peak related toDMAC or PAA. Before the membrane was used to wrap the cheese, it wasrinsed with tap water 10 times, and then rinsed with 70% Ethanol; inorder to remove residual ethanol, the membrane was kept in pure waterfor 3 h.

As seen from FIGS. 2a-2q , pure poly(amic)acid did not have anyaliphatic groups while it did possess carboxyl, amino, carbonyl andaromatic groups. Due to the two ways of ODA-PMDA interactions, carboxyl,carbonyl and amino groups showed two different environments.

Insets in FIG. 2a show the cis- and trans-forms of PAA. These twochemical environments affect proton shifts seen in NMR spectra. Theyhave an impact on structural characterization.

Even though two amino protons were observed, only one carboxyl protonwas observed; this difference is related to the fact that the carboxylproton is more isolated despite the fact that two carboxyl carbons wereobserved. However, in the case of very high amount of PAA membranedissolved in DMSO-d₆ to run NMR, the carboxyl proton was not observedeven though carboxyl protons were present; similar results were observedfor PAA-GA-SA membranes.

Further, introduction of GA to PAA resulted in the presence of protonpeaks related to carbonyl and aliphatic groups. In parallel to theincrease in GA concentrations, the peaks became sharper and morevisible. As seen from FIGS. 3a-3f , GA can give peaks between 4-6 ppmdue to the presence of double bonds. Therefore, the aliphatic protonsprovided in the Table I can be speculated as coming from GA. GA alsoshowed its presence via the alterations in the aromatic region; higherconcentrations of GA eliminate presence of the peak at about 7.74 ppmwhile the carbon peak related to that group remained same. As seen fromFIGS. 3a-3f ¹H COSY, ¹H ¹³C HSQC and ¹H ¹³C HMBC, the protons peakremained same. However, the adjacent peaks around the major PAA aromaticprotons decreased, which is a sign of GA interaction to the phenyl ringof ODA.

Amino groups did not show any change in response to GA action while thepresence of new peak at 10.65 ppm was observed in the cases ofsulfanilic acid (SA). However, ¹H ¹⁵N HSQC and ¹H ¹⁵N HMBC did not showthe presence of new amino groups: there was only one type of aminogroup. This can be speculated to mean that either SA content was notenough to be seen or prior treatment of SA with GA resulted in secondaryamino group formation. ¹H COSY reveals the presence of aromatic protonand amino proton of SA interacting each other. Therefore, it is clearthat SA chemically bonded to the PAA backbone.

Overall, GA chemically binds to the PAA backbone from phenyl ring of ODAlocated at the edges of the individual PAA polymers. Prior treatment ofSA with GA results in the elimination of primary amino groups, and madethem visible as secondary amino groups with PAA-SA-GA polymers.

NMR was also used to characterize the chemical stability of PAA-GApolymer. The polymer was kept in an air-tight flask under sun-light forover 3 years. As seen from FIG. 4, PAA polymer lost its structuralintegrity, and gave fragmentation and oxidation peaks; this wassupported by presence of multiple aromatic protons and amino protons,and loss of carboxyl proton. Besides, the adjacent peaks, particularly,around 7 ppm gave the same integral of the major peak which is a sign offragmentation of individual PAA polymers as shown in FIGS. 2a -2 q.

Example 1.2.1-Molecular Weight Characterization of PAA Polymers by NMR

Molecular weight (MW) characterization of the PAA polymers by NMR wasperformed using two approaches ¹H DOSY and T₁-relaxation times.

¹H Diffusion ordered NMR Spectroscopy (¹H DOSY) is a two-dimensional NMRtechnique which relies on the relation between molecular mass of amolecule/polymer and its self-diffusion. The technique has been shown tobe useful in determining the average molecular weight of a polymer. Itis based on the theory of the Stokes-Einstein equation. In all DOSYexperiments samples was 1.2-1.4 mg/mL in DMF-d₇unless stated otherwise.In DOSY NMR experiments (a technique giving information about theaverage molecular weight of the molecules), concentration of themolecule/polymer should be low enough (1.2-1.4 mg/mL) to avoid viscosityrelated biased results.

As can be seen from FIG. 6, which illustrates the standard graphics of¹H DOSY, Polystyrene standards at 10^(3.114) Da, 10^(4.455) Da,10^(5.236) Da and 10^(6.34) Da MWs were used to draw the standardgraphic. All standards were prepared ˜1 mg/mL in DMF-d7.

DOSY results of some PAA synthesized in the study are shown in Table Hbelow.

Polymer ¹H DOSY MW (Da) PAA-DA-GA (0.12M)  1.6 × 10⁵ PAA-pAB-GA (fresh)(0.12M) 1.22 × 10⁵ *Standard mixture 1 4.49 × 10⁵ ^(#)Standard mixture 21.01 × 10⁶ ⁺Standard mixture 3 1.44 × 10⁵ PAA-I-W-GA (0.12M) 1.76 × 10⁵PAA (0.14M) 40° C. 3.01 × 10⁵ PAA (0.16M) 1.68 × 10⁵ PDA-PAA (0.16M)1.49 × 10⁴ PAA (0.12M) 2.11 × 10⁵ PAA-IZ (0.12M) 1.78 × 10⁵ PAA (0.10M)5.28 × 10⁵ PAA (0.14M) 2.33 × 10⁵ 0.12M PAA-pAS-GA (fresh) 1.36 × 10⁵0.08M PAA (1:1.03) 40° C. 5.28 × 10⁵ 0.08M PAA-GA (aged) 1:1.03 40° C.3.41 × 10⁵ 0.08M PAA-GA (fresh) 1:1.03 40° C. 4.02 × 10⁵ 0.16M PAA(1:1.03) 3.81 × 10⁵ 0.12M PAA in 65:35 Ethanol:DMAC, 40° C. 2.61 × 10⁵0.12M PAA in 50:15:35 Ethanol:H₂O:DMAC 1.14 × 10⁵ 40° C. PAA (0.14M) 30°C. 2.33 × 10⁵ 0.12M PAA in 60:40 Ethanol:DMAC, 40° C. 1.78 × 10⁵GA-autopolymer Less than 10^(3a) GA-SA Less than 10^(3a) 0.12M PAA, 1:1,Room temperature-cleaned 1.35 × 10⁵ 0.12M PAA-GA, 1:1, Room temperature-  6 × 10⁵ cleaned 0.12M PAA-GA-SA, 1:1, Room temperature- 7.35 10⁵cleaned In Table H, *Polymer mixture 1 [23% of 10^(3.114); 58% of10^(5.236) and 19% of 10^(6.34)]; ^(#)Polymer mixture 2 [14% of10^(6.34), 2.6% of 10^(3.114)]; ⁺Polymer mixture 3 [38% of 3.11, 14% of10^(4.455), 19% of 10^(5.236), 29% of 10^(6.34)]. IZ: Carbodiimizole;1:1.03 refers to ODA:PMDA ratio; I: isoleucine; W: L-tryptophanemethylester; pAS: p-aminoscalicylic acid; PDA-PAA refers top-phenylenedianiline + pyromellitic dianhydride PAA; SA: sulfanilicacid. ^(a)Refers to the value was below lowest MW of standard, so it wasnot calculated.

¹H DOSY is a technique to identify average MW of polymer mixtures. Fourindividual polystyrene standards and three mixtures of them were used inorder to generate the standard graphic shown in FIG. 6, and evaluate theparameters of ¹H DOSY experiments. As seen from the standard graphic, ¹HDOSY has less than 0.01% uncertainty. Table 3 shows that ¹H DOSYprovides highly satisfactory results for revealing the average MW of thepolystyrene polymer mixtures.

Typically, crosslinked PAA polymers are supposed to show higher molarmasses (MS). ¹H DOSY experiments showed that even individual PAApolymers showed higher MS than glutaraldehyde (GA) crosslinked PAA.Further tests include aged GA-crosslinked PAA, (fresh) GA-cross-linkedPAA, GA autopolymers, and GA-small molecule co-polymers gave more cluesabout the size of the membranes. Among the cross-linked PAA polymers,fresh GA-PAA gave the highest value while PAA-W-GA (aged) gave thelowest MW. Since it is not possible to apply a strict control on theactivity of GA, there can be a variety of co-polymers which could begenerated from just the GA autopolymer-PAA, GA autopolymer, GA-smallmolecule copolymer, PAA-GA-PAA copolymers etc.

Comparison of different concentrations of PAA and the solvent systemsshowed that the average PAA size was not changed. However, heattreatment and ODA:PMDA ratio affected the MW. Based on ¹H DOSY dataalong with the observed viscosity, 0.12 M PAA prepared with Ethanol/DMACmixture at 40-50° C. was employed as the standard film condition for anytype of application described throughout the present disclosure.

NMR data provided additional information about the MW of polymers basedon T₁-relaxation times, which relies of spin-lattice relaxation. Due tothe fact that PAA polymers possess aromatic protons, T₁ relaxation timeswere compared in order to compare the MWs of the synthesized polymers.According to T₁ relaxation time test, heavy crosslinking by GA increasesthe MW of PAA polymers in accordance with the present disclosure.

IR Characterization—Functional groups on PAA and PAA-copolymers weredetermined with a Spectrum 65 FT-IR spectrometer [Perkin Elmer, Waltham,Mass.]. Membranes at solid-state was used to perform IR study. Theresults are tabulated in Table I.

TABLE I Effect of GA on shifts in IR functional groups Film O—H NH₂/NHC═O C—N C═C Phase 3688/3222/ 3422/3161/ 1692/1769 1352/1287/ 1352/1452/inverted- 2680 1624/1578 1306 1520/1580 PAA All of 3224/2700 3432/3164/1812/1170/ 1352/1308/ 1636/1444/ the 1578 1668 1289 1464/1526/ modified1574-1578 PAAs

Stand-alone membranes were directly used for IR-characterization; themembranes were not crushed into powder or located onto IR cards.

Functional groups on PAA and PAA-copolymers were determined with aSpectrum 65 FT-IR spectrometer [Perkin Elmer, Waltham, Mass.]. Membranesat solid-state were used to perform IR study.

FIG. 7 illustrates the IR spectrum of different PAA co-polymers. Series:1: PAA-A-GA; 2: PAA-pAB-GA; 3: PAA-PCI-GA; 4: PAA-PCI-GA (direct hood);5: PAA-DPC-GA; 6: PAA-C-GA; 7: PAA-BB-GA; 8: PAA-W-GA; 9: PAA-A-GA(direct hood); 10: PAA-A-GA (partially dissolved A); 11: PAA-pAB-GA(direct hood). Stand-alone membranes were directly used forIR-characterization; the membranes were not crushed into powder orlocated onto IR cards.

As seen from Table I, GA modification shifted the IR peaks to slightlyhigher frequencies for a majority of the PAA functional groups which isa sign of increases in mass of the polymers, which was depicted by ¹HDOSY results as GA increased MW of PAA polymers up to 5 times. Besides,abundant peaks for C═C and C═O bonds were observed while O—H and —NHshowed less peaks. Due to some groups overlapping in these polymers,characteristics of certain added groups were not observed in IRspectrometry. As seen from NMR characterization, introduction of GA andsmall molecules reveal more peaks correlated to —C═O and —C═C— groups,so it implies that the extra peaks seen are from GA and the smallmolecules. Decreases in O—H and —NH peaks could be related to the datathat shows that cross-linking with GA might be shifting the amino groupsresulting in overlapped and/or non-differentiable in IR spectra, whosespectrums are provided in FIG. 7.

FIG. 8a is NMR data for ¹H, FIG. 8b is NMR data for ¹H COSY, FIG. 8c isNMR data for ¹H ¹³C HSQC and FIG. 8d is NMR data for ¹³C NMR spectra ofthe aged GA while FIG. 8e is NMR data for ¹H, FIG. 8f is NMR data for ¹HCOSY, FIG. 8g is NMR data for ¹H ¹³C HSQC and FIG. 8h is NMR data for¹³C NMR spectra of stock GA did not show characteristic alterations ingroups.

¹H COSY showed that the interaction at 0.9-0.9 ppm, 1.45-2.43 ppm,2.47-9.65 ppm and 4.08-6.47 ppm were only seen for stock GA. Actually,the interaction at 0.9 ppm shows that the peak at 0.9 ppm of stock GAwas not seen in the aged GA.

Comparison shows that ¹H ¹³C has some differences as well such as theaged GA has more interaction at 1.2-1.7 (H) 13-35 (C) ppm and 4.6-5.2(H) −93-97 (C) ppm ranges.

Integration of the characteristic peaks in ¹H showed that agingdecreased free available carbonyl groups. GA can have different forms inaqueous solutions, some of them are shown in FIG. 9. The peaks at 12ppm, 9.6 ppm, 6.0-6.5 ppm range, 4.5-5.2 ppm range, 1.0-2.0 ppm wereaccepted as that these peaks are from hydroxyl groups, carbonyl groups,cyclic groups, the protons of double bond containing C groups andhydrogen of saturated carbons, respectively. Carbonyl group has thefunction of GA to show its cross-linking potency; that's why, itsintegration was calibrated to 1, and the rest was calculated relative tothe carbonyl integrals. For the aged integrations were obtained as 0.057(—OH), 1 (HC═O), 3 (H-cyclic), 16.1 (HC═C) and 50 (—CH₃) while theintegrations of the stock (fresh) GA were obtained as 0.04 (—OH), 1(HC═O), 2.14 (H-cyclic), 14.26 (HC═C) and 44 (—CH₃). This shows theaging decreased the percentage of free carbonyl group around 30% incomparison to stock GA. Presence of doublet C═C bonds and cyclicC-residues increased. This could be the reason of getting colored andfluorescent active PAA with aged GA in comparison to the stock GA.However, it should be mentioned that it is not required to use aged GAto get colorful and fluorescent active PAA; the stock GA can bedissolved in DMAC, followed by introduced to PAA or PAA-small moleculemixture to get colorful and fluorescent active membranes.

Example 1.3—Scanning Electron Microscopy/Optical Characterization

Characterization of the PAA membrane morphology was carried out on aZeiss Supra 55 VP field emission scanning electron microscope (SEM). Themembranes were imaged both before and after filtration. All samples werecoated with 2-5 nm gold layers for SEM imaging.

Only the membranes produced according to FIG. 1b were characterized foroptical properties. Uv-vis properties were evaluated using HP Agilent8452 spectrometry while Shimadzu RF 6000 fluorometer was utilized tocharacterize fluorescence properties. Uv-vis characterization was onlyperformed for the stand-alone films while both stand-alone membranes andtheir dissolved forms were utilized for fluorescence characterizations.

Digital images of ternary PAA membranes from FIGS. 1a and 1b . a—PAA;b—PAA-DA; c—PAA-A; d—PAA-A was incubated in 30 min at 70° C. in additionto overnight incubation; e—PAA-A similar to d but higher GAconcentration; f—PAA-A same GA concentration to e, but just incubated inroom temperature; FIG. 1a . GA was applied at different concentrationsto the PAA solutions. g-PAA-A with % 0.3 GA; h-PAA-A with % 0.9 GA;i-PAA-CA with % 0.3 GA; j-PAA-CS with % 0.3 GA; k-PAA with % 0.3 GA;l-PAA with % 0.9 GA, and m-PAA-DA with % 0.9; FIG. 1a i. n-PAA-A 3hincubation; o-PAA-A; p-PAA-C; q-PAA and r-PAA-DA. FIG. 1 a iii with 0.9%GA from 70% GA stock. s-PAA with % 0.3 GA; FIG. 1a ii. This showedwoven-like surface as shown by SEM imaging. The images “t” and “u” aresynthesized with FIG. 1 a iii with 0.35% GA concentration. In 6h, PAA-CSgave green membrane [t] which could be peeled off from glass surface,which gave gel-like structure. The gel like membrane [t] was thenphase-inverted in pure water and incubated overnight under hood [u]. Thefollowing membranes were prepared according to FIG. 1b ; v: PAA-5AS-GA,w: PAA-4AS-GA-MeOH, x: PAA-AcOH-CA-GA, y: PAA-pAB-GA, z:PAA-AcOH-Ser-GA-MeOH, aa: PAA-PCl-GA, ab: PAA-AcOH-A-GA-MeOH, ac:PAA-5AS-GA but this is just incubated in RT, ad: PAA-5AS-GA but directhood evaporation, ae: PAA-PCl-GA-MeOH [right after GA], af:PAA-MeOH-Ammonium Nitrate-GA [direct hood], ag:PAA-PCI-GA,ah:PAA-5AS-GA, ai:PAA-A-GA. Even though the images v, ac, ad and ah aremade out of PAA-5AS, 5AS content and incubation procedure affect thecolor formation; ah has the lowest 5AS concentration. aj-PAA-I-GA.

Digital images of some films from FIG. 1b are shown in FIGS. 11a-11l .All the films were prepared according to FIG. 1b , and GA concentrationwas 0.1% while PAA was 0.12 M; a: PAA phase inverted under hood; b:pAB-GA-PAA; c: W-GA cross-linking for 15 min then introduced into PAAsolution; d: pAB dissolved in DMAC incubated with GA for 15 min,followed by introduced into PAA solution; e: pAB was added to PAAsolution, followed by addition of GA; f:W-GA cross-linking for 5 minthen introduced into PAA solution; h::pAB dissolved in DMAC incubatedwith GA for 30 min, followed by introduced into PAA solution; i:W wascrosslinked with aged GA, followed by introduced into PAA; j: pAS and Wwere added into PAA solution, followed by addition of GA; k::pAS wasdissolved in DMAC, and then added into PAA solution, followed by added0.2% pAB-GA (at that moment the incubation was passed already 30 min);j: W cross-linked with fresh GA (stock 70%), followed by added into PAA.

A discussion of FIGS. 10a -10 aj and FIGS. 11a-11l follows.

As seen in FIGS. 10a -10 aj, PAA-DA gave some blue region but the restis yellowish due to the fact that high amount of GA stacked in localizedplaces because of high viscosity-related quenched stirring. Similarly,FIG. 10 ac, FIGS. 10 ah-10 ai and FIG. 11b and FIG. 11h possessed unevensurfaces. Interestingly, increased incubation time and high GAconcentration form colorful plastic like membranes in FIG. 1a , theseparameters didn't show any significant effect on formation of differentcolored membranes with plastic-like structures. However, for both FIGS.1a and 1b , treating GA with DMAC alters the formed color as well asaffecting on the other parameters such as contact angle and mechanicalstrength. For example, the membranes FIGS. 10 m and 10 r were fromPAA-DA. Even though, the membrane FIG. 10m is plastic-like transparentwhile the membrane FIG. 10r is opaque and dark-blue color withpossessing higher contact angle; top/bottom contact angles of themembranes FIGS. 10 m and 10 r were 62.35/55.7 45.3/47.3, respectively.Due to the aggressive nature of GA, it can make excessive cross-linkingin PAA solution.

Comparing FIG. 1 a i and iii for same GA concentration and incubationperiods, it was shown that pre-dissolving GA in DMAC makes it much moreactive; this can be resulted from that dissolving GA from stock in dryDMAC partially or totally altering GA microenvironment, which then mightchange binding preferences and/or rate of binding. Formation oftransparent membrane also strongly depends on the small molecule addedto the PAA solution. For example, L-Alanine added PAA membranes alwaysformed transparent plastic like membranes if a special treatment was notapplied even for FIG. 1 a.

However, L-Tryptophan methyl ester, L-Isoleucine and some other smallmolecules resulted in opaque membranes. Individual PAA, PAA-DA andPAA-CS are the ones gave distinctly different membrane formations byjust shifting the procedure, FIG. 1 a i to iii. It should be noted thatthe membranes of FIG. 1a were partially or totally formed before rinsingstep. Unless the membrane is totally formed, rinsing with water formspartially or totally opaque membranes, which can be explained with themodel proposed elsewhere. However, further drying (after rinsing step)turns the opaque membranes into transparent forms within 24 h under hoodfor relatively higher GA concentrations such as % 1; but mostly theseare brittle. For example, the membrane FIG. 10u couldn't be turned intoa transparent membrane even at 48 h incubation. This could be related tothe high DMAC content formed thicker interacted with non-solvent.

In contrast to this, it is possible to synthesize substantiallytransparent and durable membranes of FIG. 1a for all of PAA-smallmolecules even PAA-I if the GA concentration is higher 2% withpre-dissolved GA in DMAC. Using low GA concentration as 0.35% still canprovide substantially transparent and durable membranes, but theincubation time should be 12 h at room temperature and 12 h under hood.12 h incubation does not totally remove DMAC, but further rinsing doesnot cause any opaque-structure formation.

FIG. 12m is photographs of PAA-SA (warmed)-pAS-5AS-GA, FIG. 12nPAA-SA-pAS-5AS-GA, FIG. 12o PAA-SN-GA, FIG. 12p PAA-SN-pAS-GA. Colorchange of the same films can also be manipulated by heating the smallmolecule, or introducing other small molecules at very low quantity.Warming up SA before it was pre-treated with GA changed the resultantfilm color from yellow to brown while FIG. 12p has only 0.1 mg/mL pAS inaddition to FIG. 12o , but the color did changed.

Color formation in FIG. 1b is distinctly different from FIG. 1a .Pretreatment of small molecule with GA, GA condition (aged or fresh) andpresence of cross-linking quenchers are the predominant parameters whichcan be even confirmed by only FIG. 10 al. For instance, pretreatment pABwith aged GA provided blueish membrane formation while adding pABdirectly into PAA-GA mixture formed slightly maroon color membrane.Another example is that using fresh GA instead of aged GA resulted inshifting the color from green (FIG. 11i ) to yellow (FIG. 11l ).

Example 1.3.1—UV-Vis Spectra of PAA and Ternary PAA Membranes

UV-Vis spectroscopic properties of PAA membranes were evaluated todetermine the effect of small molecule and GA on formed membranes. PAAphase-inverted membranes that were processed in the hood were comparedwith the PAA that were synthesized according to FIG. 1 a.

FIG. 13a illustrates UV-vis of some of the synthesized membranes. PAA:Poly(amic)acid; A: L-alanine; GA: glutaraldehyde; 5AS: 5-aminosalycylicacid; I: L-isoleucine; pAB: p-aminobenzoic acid; W:L-tryptophan-L-methylester; pAS: p-aminosalycylic acid.

FIG. 13b illustrates ransmittance of some of the membranes synthesizedin the study. PAA: Poly(amic)acid; A: L-alanine; GA: glutaraldehyde;5AS: 5-aminosalycylic acid; I: L-isoleucine; pAB: p-aminobenzoic acid;DA; glucosamine; pAS: p-aminosalycylic acid; SN: sulfanilamide.

In FIG. 13a , there is no PAA peak from 400 to 700 nm range. The peaksare related to a small molecule being introduced to a PAA molecule. Eventhough the overall color of the membranes showed strong dependence onthe condition of GA and GA pretreatment of small molecule, UV-Vischaracterization did not provide any significant difference.

Transmittance of the membranes is important for food packaging materialapplications. All of the membranes showed over 65% transmittance between450 to 700 nm. The used membranes (i.e. PAA-I-GA, PAA-I provided goodvisibility for monitoring food conditions. However, PAA-SA-pAS-5AS-GAand PAA-SN-pAS-5AS-GA have lower % transmittance at certain wavelengthssuch as ˜510 nm and 650 nm. It should be mentioned that these are notaffecting the overall visibility of the packaged food.

Unlike UV-Vis properties, fluorescence characteristics of PAA membranesshowed strong dependence on GA condition, incubation period, GApretreatment with small molecules and the presence of methanol andethanol. However, it should be noted that optimizing the conditions arechallenging due to the fact that GA can crosslink a variety of othergroups including primary/secondary amino groups, thiol groups, hydroxylgroups of sugars and aromatic carbons.

The Fluorescence Characteristics of several films are discussed below.FIGS. 14a and 14b illustrate Rhodamine 6G standards.

FIGS. 15a-15e illustrate several spectra. The spectra seen in “a” and“b” belong to yellowish PAA-A-GA membranes while the spectrums seen in“c” and “d” belong to the greenish PAA-A-GA membrane. The spectrum “e”belongs to PAA-GA. All of the membranes were synthesized according toFIG. 1a , and standalone membranes were used during fluorescence run.Excitation wavelengths were 581 nm, 598 nm, 608 nm and 596 nm for a-dmembranes, respectively. Emission ranges were 592-648 nm, 610-698 nm,619-699 nm and 600-640 nm for a-d membranes, respectively. Absorbancewas kept below 1 for all, and during fluorescence measurementsensitivity was kept high. As it is seen, for all membranes,fluorescence intensity started with a decreasing trend, followed byincreases in the intensity. However, PAA-GA showed an increasing trendfor fluorescence intensity from the starting point, whose excitation was612 nm while the emission range was between 620 nm and 700 nm. More thanone maximum-emission peak was observed for all. The fluorescence quantumyields of these membranes were below 0.1.

FIGS. 16a and 16b illustrate fluorescence intensity vs. emission andemission wavelengths. The membrane seen in FIG. 16a was dissolved in DMFat three different concentrations as 1, 0.67 and 0.5 for spectrum “a”while 1, 0.75 and 0.5 for spectrum “b”. Excitation/Emission wavelengthswere 608 nm/619-699 nm range and 621 nm/632-700 nm range for thespectrum “a” and “b”, respectively. Similar to FIG. 16b of solidmembrane, more than one maximum emission peaks were observed. Dilutionenhanced the observed fluorescence intensity, while the dilutionsdecreased the UV-Vis absorbance of the corresponding solutions.

FIGS. 17a-17e are wavelength illustrations. In these figures 2 mg/mL pClwas introduced into 10 mL of PAA viscous solution, followed by 200 μL GAfrom aged 25% stock was introduced to the PAA-pCl solution. The mixturewas casted on glass to prepared PAA-pCl-GA membrane according to FIG. 1b. (a) Ex 485 nm/Em 486-700; (b) Ex 505 nm/Em 506-700 nm; (c)Ex 523 nm/Em524-700 nm(d) Ex 550 nm/Em 551-700; (e) Ex 602/Em 603-700 nm. The bestquantum yield was 0.2 (Ex 523/Em 524-700), for the rest was between0.17-0.19.

FIGS. 18a-18d are wavelength illustrations. In these figures 20 mg pCland 200 μL of aged 25% GA were simultaneously dissolved in 2 mL DMAC,and mixed for 5 min. The solution was then added to 8 mL PAA solution,which was mixed for 10 min before casting on the glass to prepare themembrane according to FIG. 1b . (a) Ex 598 nm/Em 599-700 nm; (b) Ex602/Em 603-700 nm; (c) Ex 657 nm/658-700 nm; (d) Ex 621 nm/Em 620 nm/Em621-627 nm. The best quantum yield was 0.1 (Ex 598 nm/Em 599-700 nm).

FIGS. 19a-19d are wavelength illustrations. In these figures 20 mg pABand 200 μL of aged 25% GA were simultaneously dissolved in 2 mL DMAC,and mixed for 10 min. The solution was then added to 8 mL PAA solution,which was mixed for 10 min before casting on the glass to prepare themembrane according to FIG. 1b . (a) Ex 485 nm/Em 486-650 (but shown486-526 nm); (b) Ex 523 nm/Em 524 nm-700 nm (shown 524-600 nm); (c)range between 535 nm to 600 nm of b; (d) Ex 619/Em 620-700 nm. Series 1always refer to the thicker PAA-pAB-GA while series 2 depicts thethinner PAA-pAB-GA (aged). The best quantum yield obtained was 0.1 (Ex485 nm/Em 486-650 nm of Series 2).

FIGS. 20a-20d are wavelength illustrations. In these figures 10 mg I wasdissolved in 200 μL of aged 25% GA, which was then vortexed for 10 min.I-GA mixture was then introduced to 10 mL PAA viscous solution, andmixed for 10 min, followed by casted on glass surface to preparePAA-I-GA membrane according to FIG. 1b . (a) Ex 567 nm/Ex 568-700 nm;(b) Ex 587 nm/Em 588-700 nm; (c) Ex 590/591-700 nm; (d) Ex 601 nm/Em602-700 nm. The best quantum yield obtained was 0.08 (Ex 590/591-700nm).

FIGS. 21a-21d are wavelength illustrations. In these figures 20 mg pASand 200 μL of aged 25% GA were simultaneously dissolved in 2 mL DMAC,and mixed for 10 min. The solution was then added to 8 mL PAA solution,which was mixed for 10 min before casting on the glass to prepare themembrane according to FIG. 1b ; (a) Ex 485 nm/Em 486-600 nm; (b) Ex 523nm/Em 524-600 nm; (c) Ex 550 nm/Em 551-600 nm; (d) Ex 598 n/Em 599-700nm. The best quantum yield obtained was 0.08 (Ex 523 nm/Em 524-600 nm).

FIGS. 22a-22d are wavelength illustrations. In these figures 10 mg I wasdissolved in 200 μL of aged 25% GA, which was then vortexed for 10 minand 2 min heated at 70° C. sequentially. I-GA mixture was thenintroduced to 10 mL PAA viscous solution, and mixed for 10 min, followedby casted on glass surface to prepare PAA-I-GA membrane according toFIG. 1b . (a) UV-Vis of the solid membrane; (b) Ex 523 nm/Em 500-550 nm;(c) Ex543/Em 544-610; (d) Ex598/Em 590-610 nm. The best quantum yieldobtained was 0.1 (Ex543 nm/Em 544-610 nm).

FIGS. 23a-23e are wavelength illustrations. In these figures 10 mg DAwas dissolved in 200 μL of aged 25% GA, which was then vortexed for 10min. DA-GA mixture was then introduced to 10 mL PAA viscous solution,and mixed for 10 min, followed by casted on glass surface to preparePAA--GA membrane according to FIG. 1b . (a) PAA-DA-GA (aged) UV-Vis ofthe solid membrane; (b) Ex450/Em451-500; Ex487/Em488-510; (c)Ex523/Em524-590; (d) Ex601/Em602-700. Ex543/Em 544-610. The best quantumyield obtained was 0.24 (Ex487/Em488-510) while at the other excitationsquantum yields were observed between 0.1-0.17.

Then a series of films were synthesized to test the fluorescenceproperties with Synchronous Fluorescence Spectroscopy. PAA-pAS-GA,PAA-pAB-GA, PAA-W-GA and PAA-W-GA combined with GA treated pAS.

In FIG. 24, Series 1-4: (1) PAA-pAB-GA (aged); (2) PAA-pAS-GA(aged)-purplish; (3) PAA-pAS-GA (aged)-greenish; (4) PAA-W-GA(aged)-reddish.

FIGS. 25a-25c are wavelength illustrations. In these figures PAA-pAB-GA(aged) greenish. First, pAB was cross-linked with aged GA in DMAC for 20min, followed by introduced to PAA solution which was then stronglymixed for 10 min. The solution was then casted on glass, and incubatedat room temperature for 6 h, followed by incubated under hood for 6 h.Then, the resulted standalone membrane was rinsed with nano-pure water.Experimental conditions for fluorescence was as described follow; roomtemperature, quartz fluorescence cuvette, standalone membrane itself,1.5 slit width, excitation filter auto, emission filter auto, atsynchronous mode, delta 0 nm, run mode was slowest (30 nm/min), start220 nm, stop 700 nm. The instrument was Cary Eclipse run with Eclipsesoftware. PAA-pAB-GA, slit 1.5, speed normal, delta 0.1 (b) and 1 nm(c).

FIGS. 26a-26g are wavelength illustrations. In these figures Synchronousfluorescence of modified PAA and Rhodamine B. (a) PAA-pAS-GA (purplish);(b) PAA-pAS-GA (greenish); (c) PAA-W-GA (reddish); (d) PAA-W-pAS-GA(purplish); Rhodamine B with delta 0 nm (e) and 10 nm (f); (g) formedPAA-pAS-GA membrane was dissolved in DMF, and 0.1 mg/mL 2-aminopyridinewas added to the dissolved PAA-W-pAS-GA, which was then casted on glassfor evaporation mediated membrane formation. In all cases experimentswere performed for standalone membranes in quartz fluorescence cuvetteat room temperatures. The fluorimeter was set to auto mode forexcitation filter and open-mode for emission filter while scanned regionwas kept between 250 nm and 700 nm under slowest run which was 30 nm/minfor the instrument. Slit width for excitation and emission were selectedas 1.5 for “a”, “c”, “d” and “g” while they were set to 2.5 for “b”, “e”and “f”. Delta was selected 0 nm if not specified otherwise. All of themembranes were prepared according to FIG. 1b . To make PAA-pAS-GA green,pAS and GA were simultaneously dissolved in DMAC where GA cross-linkedpAS for 20 min. Then, mixture of PAA-pAS-GA was introduced to viscousPAA solution containing 0.5 mg/mL pAS for 20 min mixing which was thencasted on glass to form membrane according to FIG. 1b . Reddish PAA-W-GAwas synthesized through introducing 20 min GA-crosslinked W into viscousPAA solution.

In the above reference figures, increasing delta from 0 to 10 nm,improved fluorescence intensity was observed as expected sincepossessing too close excitation and emission wavelengths causesdecreases in fluorescence intensity. However, for the membranesincreasing delta from 0 to 1 nm, disrupted the observed fluorescenceintensity. This was most probably from that over-load of fluorophore inthe medium; fluorophores within the membranes can show FRET which thendiminished the observed fluorescence intensity. This idea was supportedthrough adding more diverse fluorescent active molecules in themembrane. Introduction of pAS into PAA-W-GA decreased the observedfluorescence. Similarly, introduction of GA cross-linked pAS intoPAA-pAS diminished the fluorescence intensity. PAA-pAS-W-GA did notprovide enough fluorescence intensity when the slit width was 1.5; thehighest peak was around 0.5 (not shown). However, dissolvingPAA-pAS-W-GA in DMF, and introduction of 2-aminopyridine to the membranegave a visible spectrum even at 1.5 slit width. However, if the PAAmembrane was used without dilution, the intensity was not visible at 1.5slit width.

As seen from FIGS. 14a and 14b , Rhodamine 6G provides very smoothexcitation and emission curves while the PAA membranes did not provideany smooth curves. PAA films can be synthesized as fluorescence active,but the best quantum yield was obtained around 0.1 leaving 10 nm gapbetween excitation and the starting emission wavelengths. These figureswere used to compare two things; (i) that the dyes can be introducedinto the PAA membrane, where their presence is strong and similar to theoriginal dyes molecules, (ii) FIGS. 14a and 14b represent the graphicalillustrations of absorbance and emission values of various PAA film andthese were used to calculate the quantum yields of the dye-modified PAAmembranes.

Besides the selection of the emission range, the conditions must becontrolled for reproducible results:

(1)Glutaraldehyde must be aged to obtain fluorescence active membranes,which was determined experimentally in the study. NMR spectroscopy canbe used to keep the best conditions. (2) The presence of methanol endethanol during membrane formation prevents fluorescence active membraneformation, which can be overcome with addition of water. (3) Humidityand longer incubation at room temperature disrupts UV-Vis andFluorescence properties of PAA membranes. The humidity was not measuredexperimentally. (4) Care required during the addition of GA into thePAA-small molecule mixture. It should be drop by drop; sudden additionof high amount of GA prevents membrane formation.

Due to possessing unique properties and low cost, conjugated polymersare preferred as a sensor support material or direct sensing agent fromelectrical to optical sensors.

pAS was introduced into different polymers, and its fluorescent activitywas shown dependent on the chemical and physical properties of thepolymer, including ionization of side groups within the polymer, allowedvolume and polarization properties of the groups within the molecules.Above, pAS is becoming part of the membrane itself. So, it is quitenormal that fluorescence characteristics of pAS will show change.Besides the environment itself, the solvent also possesses strong effecton excitation and emission spectra, and quantum yield as well. Forexample, pAS itself gives only one emission in aprotic solvents, itgives more than one peak in protic solvents.

PAA itself is inherently not fluorescence-active, but introducing sidegroups to PAA was shown as a method to add fluorescence character to PAAsuch as grafted PAA with toluene-2,4-diisocyanate and straight alkylchains showed fluorescence properties. Maximum absorption and excitationwavelengths of the high purity Rhodamine 6G in anhydrous ethanol wereobtained at ˜527 nm and ˜550 nm, which refers to the stoke shift is over20 nm which matched the literature. This shows there was only onefluorophore in the medium, and the working conditions only allowedpossessing one broad peak. However, this was not valid for thestand-alone membranes and the dissolved membranes due to the fact thatthe membranes were composed of more than one fluorophore. As detailed inthe examples below, GA can cross-link individual PAA polymers as well asit can cross-link PAA-small molecule and small molecule-small molecule,which can allow possessing fluorophore. FIGS. 26a-26g depict synchronousfluorescence run of Rhodamine B and PAA-pAB-GA, PAA-W-GA (reddishmembrane), PAA-pAS-GA, respectively. SRB, as expected, provide a sharppeak which depicts that SRB in anhydrous ethanol has only onecharacteristics excitation and emission peaks while PAA-pAB-GA andPAA-pAS-GA have more than one.

As seen from FIGS. 26a-26g , when there is 0 nm difference (delta),under same conditions, between excitation and emission wavelengths forSRB, the fluorescence intensity is lower than the one in the case thedelta was 10 nm. However, this situation is distinctly different for themembranes.

As seen from the FIGS. 26a-26g , when the delta is 0, the fluorescenceintensity is the highest while the intensity decreased 400 times whendelta was 1 nm. When the delta was selected 0.1 nm, the intensitydecreased 20 times in comparison to the case of delta was 0 nm.

Quantum yield of a fluorophore is more influenced by the environment incomparison to molecular extinction coefficients. Fluorescence quenchingis the process of reduction in fluorescence quantum yield, which can beresulted from collisional quenching and/or occurrence of vibrations ofnon-fluorescent ground-state species. Self-quenching is, also, a commonproblem seen in fluorescence, which is arisen from over-load offluorophores presence. Fluorescence resonance energy transfer (FRET) isa fluorescence technique in which emission of a fluorophore is absorbedby another fluorophore as the excitation. The technique isdistance-dependent, and the yield of FRET is proportional to thedistance, which makes it sensitive.

FIG. 1b films can provide fluorescent active under strictly controlledconditions. However, current results are not allowing them to be used asa sensor material for FRET applications due to the fact that extensivefluorophore presence results in loss of quantum yield. However, studiesrelated to metals and oxygen concentrations, the synthesized fluorescentactive membranes can be a candidate for metal and oxygen sensors.

FIG. 27 illustrates excitation data for several films. Specifically,Series 1-6, respectively; PAA-Sulfanilamide-GA (Ex-Ee=1 nm);PAA-Sulfanilamide-GA (Ex-Em=5 nm); pAS in PAA (Ex-Em=5 nm); pAS in PAA(Ex-Em=10 nm); pAS+W in PAA (Ex-Em=5 nm); W in PAA (Ex-Em=5 nm).

FIGS. 28a-28c illustrate excitation and wavelength data. Specifically,UV-vis, Synchronous fluorescence of Rhodamine 6G (R6G) embedded PAAmembranes. (a) UV-vis of PAA-R6G, (b) Synchronous fluorescence ofPAA-R6G and (c) Emission of PAA-R6G.

Even though UV-vis gave peak at 570 nm, the best quantum yield wasobtained for Ex 533 nm. While R6G in ethanol solution as shown in FIGS.14a and 14b provides very smooth emission curve, R6G in PAA showsrepeating ups and downs. This could be related to that R6G within thePAA might have different microenvironments where R6G behaves differentthan how it behaves in solution. The obtained quantum yield for PAA-R6Gvaries between 0.78 and 0.82; keeping 90% of quantum yield in comparisonto pure R6G can make this type of membranes usable for fluorescentlabeled membrane applications. Since, the membrane was rinsed withexcess water, it can be said that there was no fluorescence come fromR6G stays on the membrane.

1.4 Solvent Resistance Properties

Film lengths and widths were 2.5 cm while thicknesses were between0.00128-0.00512 cm. The following buffers were used; 50 mM Acetate (pH4.5) buffer, 50 mM PBS buffers (pH 6.8/7.0/7.4), 50 mM (pH 8.0) Tris-HCland 50 mM (pH 9.6) carbonate buffer. All the buffers were prepared fromtheir salts and pH was adjusted with 1M HCl and/or 1M NaOH. Glacialacetic acid, 30% hydrochloric acid, 100% sulfuric acid, 1 M sodiumhydroxide and 37% NH4. The following organic solvents were employed forsolubility testing”: ethanol, methanol, tetrahydrofuran, hexane,ethylacetate, dimethyl formamide, dimethyl acetamide, dichloromethane,p-xylene, aceticanhydride, dimethyl-sulfoxide and acetone. The solventresistance properties of various films is discussed in the data below.

PAA films are soluble in complex media such as protein and carbohydratecontaining media and polar organic solvents. Therefore, it is essentialto show the material will not live a problem of dissolvation duringcontact to food and possible chemical contaminants or the chemicals fromfood-itself.

Solvent resistance of the synthesized PAA membranes showed closerelation to mostly GA condition and the small molecule. The findings arelisted as below.

Aged GA made the membranes soluble for all small moleculesPAA-copolymers in the case of FIG. 1 bi/ii in strong organic polarsolvents include DMF, DMAC, DMSO and buffers pH over 7.4. However, FIG.1b ii membranes showed different character such as L-Tryptophan methylester individually, or the other amino acids combined with sulfanilicacid make the membranes non-soluble in DMAC and all the buffers testedin the study.

Fresh GA made the membranes of the small molecules PAA co-polymerssynthesized according to FIG. 1b non-soluble in all the solvents testedhere except the membranes containing pAS, pAB, 5AS, PCl over 1 mg/mL.

Sulfanilic acid (SA) advanced solvent resistance character of all themembranes synthesized according to FIG. 1b . However, extra-GA must beadded to the PAA solution prior to SA addition.

Tryptophan methyl ester-PAA-GA copolymers formed non-soluble membranesin the cases of that GA was fresh; methanol addition is required, or GAaddition to the PAA solution could be beneficial to make the membranesnon-soluble.

Membranes synthesized according to FIG. 1 a except PAA-A-GA were solublein strong polar organic solvents include DMAC, DMSO, DMF, and thebuffers include 50 mM pH 8.0 PBS. Also, the most aggressive solvent forthe most resistant membranes were ammonium-hydroxide.

Solubility of all the membranes did not show dependence on pH as shownin the figures and discussed below.

FIG. 29 shows the solubility of ternary PAA membranes in basicsolutions. Solubility is not related to pH, because pH 4.5 50 mM Acetatebuffer, 1M NaOH, 1M HCl, Glacial acetic acid, pH 8.00 50 mM PBS buffer,pH 6.0 1M PBS did not dissolve the membrane. But 29% NH₄OH totallydissolved the Ile-PAA-GA membrane in less than 1 h under no agitation.This is important because ammonia is produced during food-purification.

Color changes of ternary PAA membranes in response to alcohol exposureis shown in FIG. 30. Color change related to exposure to alcohol usingternary PAA membranes. Ile-PAA; normally the membrane is greenishyellow. Alcohol turns the color into chestnut color as it was shown forEthanol, Methanol and Ethylene Glycol.

Color changes of PAA membranes in response to alcohol treatment is shownin FIG. 31. All of the membranes change their color differentintensities of red in response to alcohol treatment. Color change isaccompanied with new peak formation in UV-Vis Spectrum. While peakchange in response to Methanol and Ethylene Glycol is morecharacteristics at around 520-550 nm, the new peak formation is lesscharacteristics at around 560 nm and 250 nm. Speed of color changes isdependent on the membrane composition. In the case of L-isoleucineenhanced membranes, the color change becomes visible in less than 6 hwhile L-alanine and p-aminosalicylic acid conjugated PAA membranes showthe color change within 12 h and 120 h, respectively. The synthesizedhybrid PAA films can determine the onset of alcohol (or microbialdegradation) production. The intelligent properties of the PAA wereassessed by monitoring changes in reversible and irreversible propertiesof the membranes. Changes in voltage and color in response to microbialdevelopment and/or food decomposition would provide real-time monitoringof food condition. Color changes by alcohols are important findings forintelligent food packaging material of the synthesized membranes.Ethanol is the most common byproducts of microbial development, and canbe seen from FIG. 31, color changes can be seen with bare eyes and doesnot need to be monitored using any sophisticated tools.

Color change in response to exposure alcohol is achieved in all types ofPAA films, but when the membrane is very dark (low transparency), thechange takes a comparatively longer time. Among the membranes, Ilecontaining membranes gave the response fastest; while Ile enhancedPAA-GA membrane gave color change within 30 min, pAS enhanced PAA-GAmembrane required 2-4 h to give color change.

Color changes in response to alteration in environmental conditions canbe further advanced with pH-dependent dyes. Here bromophenol blue (BPB)was simply tested, with the results shown in FIG. 32.

In FIG. 32, pH dependence of bromophenol blue supported PAA ternarymembrane. PAA-BPB-GA (aged) was prepared according to FIG. 1b . All ofthe membranes were 4 cm×2 cm×0.05 cm (length/width/thickness). (a) 50 mMpH 6.00 PBS; (b) 50 mM pH 6.5 PBS; (c) 50 mM pH 7.1 PBS; (d) 50 mM pH7.6 PBS and (e) 50 mM pH 8.0 PBS buffer. Color changes started within 30min for c, d and e vials while color change was seen for a and b for upto 6h (test was completed in 6h). End of 6h incubation, the membraneswere removed from the vials, and no visible color change was seen for aand b (f and g, respectively) while the color has changed for c, d and e(h, f and k, respectively). The membranes were also tested for 50 mM pH4.5 Acetate buffer, and no color change was observed.

Further color change is shown in FIG. 33. In FIG. 33 at the end of 6hincubation, PAA-BPB-GA (aged) membrane was taken out from the vial. Asit is seen, the top part kept its yellowish color, because the part wasnot submerged into the buffer, while the rest of the membrane was turnedinto pale greenish form.

This section was to entrap BPB within PAA-GA, and release the BPB intothe medium when there is a change in pH and/or ionic strength; when thePAA is treated with aged GA it doesn't have insoluble form for harshenvironment such as high pH, strong organic solvents and so on. Thecolor change of the membrane itself might be resulting from certainamount of BPB crosslinked to PAA. Actually, using fresh GA can advanceBPB cross-linking to PAA, which could also a possible way of monitoringpH changes.

1.5 Mechanical Characterizations

The mechanical strengths of the membranes were determined using Instron®Tension Testers run by Merlin Project Software (Norwoon, Mass.). Thestrength tests were evaluated using maximum load, tensile strength andmodulus of elasticity.

Mechanical properties of food-packaging materials are a parameter inassessing their relevance as packaging materials. This is becausepackaging materials must protect the containment against possibleexposure to physical pressures during transportation and storage.Maximum load, modulus elasticity, break elongation and tensile strengthare common parameters to evaluate mechanical properties of a packagingmaterial. Different approaches have been applied to advance theproperties such as combination of biopolymers and chemicals, compositesor nanomaterials (i.e. chitosan and graphene, whey protein-zein).

TABLE J illustrates Mechanical properties of FIG. 1a Films MaximumModulus Break Tensile load elasticity Elongation Strength Film-Type (kg)(MPa) % (MPa) Composite^(a) 12.75 180 24.3 13.79 PAA-C-GA^(b) 5.13406.89 13.4 32.41 PAA-A-GA^(b) 3.86 1096.55 10.9 24.14 PAA-GA^(c) 1.8113.79 29.6 7.07 PAA-CS-GA 7.39 37.24 139.5 6.21 PAA-G-GA 6.08 45.52 83.18.97 PAA-W-GA 2.95 31.03 42.9 4.14 PAA-I-GA 5.67 24.14 181.3 8.97PAA-R-GA 4.68 26.89 79.8 6.89 PAA-K-GA 6.21 35.86 73.9 9.66 PAA-T-GA2.31 164.14 28.8 1.45 PAA-E-GA 5.67 58.62 67.9 8.28 PAA-CA-GA 10.6186.89 115.4 15.86 PAA-DA-GA 6.4 121.3 7.2 11.73 PAA-GA^(d) 8.62 485.5238.8 43 .45 PAA-GA^(e) 9.03 232.41 63.6 45.52 PAA-GA^(f) 12.47 737.41126.4 63.49 PAA-A-GA^(h) 9.48 821.38 8.8 28.97 PAA-S-GA^(h) 5.72 697.2430 43 .45 PAA-A-CS-GA 4.04. 72.41 8.56 6.21 PAA-C-GA 14.51 920 48.373.79 All of these films were at 0.18M PAA concentration, but some ofthe membranes were at 0.20M concentrations. ^(a)PAA-GA was casted ontoglass, and then non-GA treated W was added onto the PAA-GA. ^(b)GAtreated PAA-C incubated overnight after being casted on the glass;3-week old membrane. ^(c)GA treated PAA incubated overnight after beingcasted on the glass; 2h old membrane. ^(d)GA was first diluted in dryDMAC and then applied to PAA solution. PAA membranes were casted onclean glass and incubated overnight. After incubation, DMAC waseliminated from the membranes by soaking them in pure water. Thethickness of PAA membrane is important for its modulus elasticity and %break elongation; ^(e)while thicker one has higher (4) break elongation,its modulus elasticity is lower [i.e. thicker one has %63.6 and 273.79MPa while one was %38.8 and 485.2 MPa. Altering the incubation time wasalso an important factor as seen in ^(f)which has lower incubation time.^(h)GA was dissolved in dry DMAC and then directly applied to PAA-Asolution. The length and width of all membranes were 2.52 cm whilethickness of the membranes was between 0.025-0.1 mm.

Maximum-load bearing capacities of the membranes mostly showed relationwith the thickness of the membrane. For example, PAA-CA, Compositemembrane, PAA^(f), PAA-CS were relatively thicker than PAA-T, PAA-A^(b)and PAA-R which showed lower maximum-load bearing potential. However,PAA-C, PAA^(c) and PAA^(th) which were relatively thinner but couldwithstand high load, which is a sign of how small molecule affects themembrane's mechanical properties. Modulus elasticity and breakelongation were dependent on the procedure and the small moleculecombined with PAA. L-Cysteine and L-Alanine were by far best membranesin FIG. 1a . In terms of L-alanine, two different concentrations (1mg/mL and 2 mg/mL) were tested; we however found that the concentrationwas not so important ion but the thickness of the crystal L-alanineresidues applied on the membrane makes a difference in terms of modulusof elasticity. Larger crystals make the membrane weaker. However,manipulations in the procedure results in alterations of the mechanicalproperties.

New membranes were prepared according to FIG. 1b , and the mechanicalresults are discussed below.

TABLE K Mechanical properties of FIG. 1b films Tensile Break BreakModulus Maximum strength strength, Elongation elasticity Film Type load(kg) (MPa) (MN) (%) (Mpa) Combined^(1, 2) 5.25 26.89 24.68 3.4 1043.87PAA-A^(1, 3, 4) 3.13 47.57 22.27 6.1 1750.58 PAA-A^(1, 3, 5) 2.54 19.316.55 2.2 930.79 PAA^(1, 3, 6) 4.49 68.26 41.85 11.6 1872.62PAA^(1, 3, 7) 4.13 62.74 19.72 8 2244.93 PAA^(1, 8) 2.86 86.18 30.3416.4 1552.69 PAA^(1, 3, 4, 8) 2.18 66.19 41.58 23.8 1101.51PAA-CA^(1, 3, 4, 8) 4.13 62.74 55.85 24.7 1221.06 PAA-A^(4, 9, 10, 11)2.9937072 45.51724138 44.62068966 61.7 235.862069 PAA-A^(9, 10, 12)2.3586784 35.86206897 21.5862069 11.2 961.3793103 PAA^(7, 9, 13)2.79412672 84.82758621 65.65517241 13.8 2639.310345 PAA-A^(4, 9, 10, 14)3.9916096 30.34482759 27.86206897 9.1 1038.62069 PAA-A^(7, 9, 10, 14)1.8597272 57.24137931 47.37931034 8.4 2062.068966 PAA-A^(7, 9, 10, 11)1.7236496 52.4137931 1.103448276 11.9 1678.62069 PAA-A^(7, 9, 10, 15)3.9008912 29.65517241 29.44827586 7.4. 1031.724138 PAA-A^(7, 9, 10, 15)4.3544832 33.10344828 27.31034483 8.6 866.2068966 PAA^(8, 9, 11)2.2226008 33.79310345 25.86206897 6.7 1193.793103 PAA-A^(7, 16, 17)2.5854744 39.31034483 34.89655172 6.3 1382.758621 PAA-A^(7, 13, 18)4.4905608 33.79310345 33.03448276 14.6 1264.137931 PAA-A^(7, 8, 18)1.9504456 29.65517241 27.24137931 4.9 1400 PAA^(7, 8, 19) 3.220503248.96551724 41.31034483 14.2 1397.931034 PAA^(7, 8, 19) 3.4019451.72413793 48.13793103 29.4 1823.448276 PAA- 2.1772416 65.5172413835.86706897 9.7 2185.517241 DC^(8, 9, 20, 21) PAA-A^(8, 9, 20) 2.222600866.89655172 48.27586207 5.8 2762.068966 PAA-W^(8, 9, 20) 1.496853646.20689655 42.48275862 9 1848.275862 PAA-W^(8, 9, 20) 1.769008853.79310345 43.31034483 12.7 1880 PAA-W^(8, 9, 20) 2.9029888 55.1724137949.79310345 37.4 1615.172414 PAA-BB^(8, 9, 22) 2.26796 68.9655172458.68965517 40.9 2177.241379 PAA-DA^(8, 9, 20) 2.0865232 63.4482758610.48275862 30.4 1725.517741 PAA- 0.8164656 31.03448276 −12.2758620711.8 1315.172414 PCl^(8, 9, 23, 24) PAA- 2.4493968 46.8965517232.48275862 6.6 1368.275862 PCl^(8, 9, 23, 25) PAA-pAS- 6.25 95.14788.942 58 4101.62 SA (4 mg/mL)^(26, 27) PAA-T-pAS- 4.8 48.95 47.44 63.31793.33 SA^(26, 27) PAA^(26, 27, 28) 3.22 48.95 48.95 5.7 2255.28PAA-SA^(26, 29) 3.26 33.1 31 63.8 1245.88 PAA-pAS- 3.49 35.16 31.44 37.31531.3 SA-A^(26, 29) PAA-pAS- 3.76 38.61 31.78 27.1 1490 W²⁶PAA-SA^(26, 30) 9.66 85.49 68.12 10.4 2590 PAA- 4.35 32.6 33.1 18.4 1530SA^(26, 30, 31) PAA-IZ²⁶ 4.94 30.34 24.2 51.6 1170 PAA- 3.99 30.34 23.9954.7 1400 SA^(26, 28, 30, 31) PAA-SA-SN- 5.3 37.4 37.4 45.3 2367pAS^(26, 28, 30, 31) PAA- 5.6 91.2 84.76 57 3850 SA(4 mg/mL)- pAS-5AS-GA^(25, 26, 28) All GA concentrations were betweenn0.035-0.1% if nototherwise mentioned. In the cases of Sulfanilic acid (SA), GA wasdirectly added to the SA for pre-crosslinking, flowed added to the PAAsolution. ¹PAA concentrations were 0.18M with FIG. 1b; ²Viscous PAA wascasted on Glass, followed by 0.1 mg/mL GA added PAA introduced on top ofthe already casted PAA; ³MeOH was directly added to the system rightafter GA addition, or GA-small molecule addition; ⁴40 μL/mL MeOH added;⁵80 μL/mL MeOH added; ⁶10 μL/mL MeOH added; ⁷20 μL/mL MeOH added; ⁸FIG.1b ii; ⁹PAA concentration is 0.16M; ¹⁰FIG. 1b i; ¹¹Glass-surface wasrinsed with dry DMAC; ¹²10 min incubation at 72° C.; ¹³4 mg/mL imidazolewas used as cross-linker; ¹⁴Glass-surface is rinsed with dry MeOH;¹⁵Glass-surface and membrane surface were rinsed with DMAC; ¹⁶FIG. 1aiii; ¹⁷Glass-surface and membrane surface were rinsed with MeOH;¹⁸Glass-surface and membrane surface with EtOH; ¹⁹Glass-surface wasrinsed with EtOH; ²⁰20 μL/mL MeOH was added to the PAA solution beforecross-linker addition; ²¹4 mg/mL Diphenolcarbazide [DC]; ²²4 mg/mL2-Benzylbenzoyl; ²³4 mg/mL 4-amino-2-chlorobenzoic acid; ²⁴The castedsolution directly incubated under-hood for overnight; ²⁵4 h incubationin room temperature, and then incubated under-hood for overnight;²⁶0.12M PAA; ²⁷4 months old; ²⁸PAA prepared in 35% DMAC in Ethanol;²⁹1-3 days old; ³⁰7-10 days old; ³¹GA dissolved in DMAC was added inaddition to GA in water. In Table K, BB refers to 2-benzylbenzoyl.

Among the synthesized films, sulfanilic acid supported membranesprovided the highest mechanical property. Increasing the concentrationof sulfanilic acid from 2 mg/mL to 4 mg/mL improved the advancedmechanical properties by up to 3 times as shown in Table K. Among thesmall molecules incorporated to PAA, sulfanilic acid is the only smallmolecule containing —SOOOH group, this could be the main reason of whysulfanilic acid improved mechanical properties. The compound readilyforms diazo compounds and is used to make dyes and sulpho-drugs.

1.6 Contact Angle Characterization

Pure-water contact angles of PAA membranes were tested with CAM ContactAngle Meter [KSV Instrument, CT] run by CAM100 image recorder software.

Contact angle is the parameters typically used to evaluatehydrophilicity of food-packaging materials. It provides information onthe tendency of the material to absorb water. A good contact angle,which refers to over hydrophobic range (i.e. over 65°), cansubstantially eliminate water-vapor absorption that may triggermicrobial development on or within the packaging material.

TABLE L Contact angle of PAA membranes Procedure of Procedure ofMembrane FIG. 1a Membrane FIG. 1b Type Top/Bottom Type Top/Bottom PAA-GA55.87/51.27 PAA-GA^(b) 61.73/74.66 PAA-CS- 62.07/53.6 PAA-PCl-GA80.54/72.10 GA PAA-DA- 62.35/55.7; PAA-pAB-GA 91.49/73.63 GA 45.3/47.3PAA-CA- 87.26/95.16; PAA-AN-GA^(c) 57.74/59.21 GA 81.09/79.77* PAA-K-GA47.81/54.89 PAA-pAB- 73.45/50.75 GA^(d) PAA-A-GA 80.88/57.02; PAA-CA-65.91/66.78 56.58/63.7* AcOH-GA PAA-W-GA 47.05/65.32 PAA^(e)-AcOH-88.19/80.56 GA PAA-R-GA 44.03/51.54 PAA^(f)-AcOH- 82.84/79.88 GAPAA-C-GA 57.07/41.89 PAA^(g)-AcOH- 80.98/78.43 67.37/51.2* GA PAA-T-GA63.91/84.67 PAA-GA^(g) 80.23/79.20 PAA-I-GA 47.23/55.65 PAA-GA^(h)62.20/66.25 PAA-G-GA 66.53/69.1; PAA-A-pAS- 75.60/74.20 65.11/62.5*GA^(h) PAA-E-GA 78.78/59.09 PAA-I-pAS- 77.25/78.30 GA^(h) PAA-S-GA97.33/62.48; PAA-S-GA 52.62/60.53 74.27/61.5* Composite^(a) 95.27/80.7PAA-SA-pAS- 82.56/84.3 GA In Table L * refers to 0.5-1% GA concentrationand overnight incubation. The remaining membranes were consistent with0.1-0.2% GA concentration and 6 h incubation: ^(a)GA treated PAA castedon the glass, and then non-GA treated PAA-W and PAA-CA was added ontothe PAA-GA and left 6 h incubation. ^(b)membrane was phase-invertedunder hood; ^(c)AN refers to Amonium Nitrate; ^(d)Formaldehyde was usedfor cross-linking; ^(e)20 μL/mL olive oil; ^(f)40 μL/mL olive oil;^(g)100 μL/mL olive oil; ^(h)0.12M PAA. Standard deviations out of3-runs were less than 6% for all membranes developed.

Contact angle of the membranes synthesized according to FIGS. 1a and 1bdid not indicate a substantial difference.

For FIG. 1a membranes, when the membranes includes a shiny surface withhaving amorphous inner part gave the highest contact angle. However, themembranes containing L-lysine (K) and L-arginine (R) gave the lowestcontact angle even though they were fully amorphous; these K and Rcontaining membranes were somewhat porous.

For FIG. 1b membranes, adding acetic acid to PAA before introducing GAadvanced the contact angle from 65 to 88, but increasing olive-oilconcentration into the PAA decreased the observed contact angle.Typically, oil is thought of as hydrophobic; probably acetic-acid makingoil causing pore-opening which results in enhances water-membraneinteraction. However, adding oil into the PAA without acetic acid, itadvances the contact angle. Among FIG. 1b membranes, p-aminobenzoic acid(pAB) containing glutaraldehyde (GA) treated PAA membranes gave thehighest contact angle; this could be related to that GA eliminate freeamino-groups on pAB, which decreases hydrophilic properties of PAA.

In all cases, the present films contain sulfanilic acid,p-aminosalicylic acid and glutaraldehyde The obtained contact angle wasover 65°, thus, the small molecule incorporated within the PAA film canserve as good-packaging materials due to the contact angle data.

1.7 Electrochemical Characterization

Four-probe and Ohm meters were utilized for characterization of theelectronics properties of the membranes, as shown in FIG. 34. In thisfigure a Jandel-brand four-point probe is shown. The technique is widelyused to measure resistivity of thin conducting layers. In this system,current and voltage were measured simultaneously. The system has twocurrent and two voltage probes; it gives information about proberesistance, contact resistance and semiconductor resistance. The resultsobtained from the four-probe was more accurate than simple voltmeterreading because the contact resistance is negligible in the four-pointprobe systems.

According to 4-probe and ohmmeter, none of the films was found to beconductive. The multimeter can go up to 200 MΩ and the scale was notsufficient to measure the resistance of the membranes, therefore themembranes were accepted as insulators.

Then the films were utilized as support material for gold-coating, ashshown in FIGS. 35a-35c . FIGS. 35a-35c include digital images ofE-beamed gold on PAA ternary films and Whatman® paper. As seen from “a”and “b”, 100 nm gold layer was coated on two different PAA membranes viaE-beam under the current of 0.104 A. Similarly, 100 nm gold layer wascoated on Whatman® paper (c) under same conditions.

Four-point probe resistivity of gold e-beamed PAA ternary membrane andWhatman® paper. FIG. 36a shows the current-voltage graphic of the coatedgold from 4-probe test at 4-different sections on the paper; FIG. 36bshows current-voltage graphic of the coated gold from 4-probe test onPAA-SA and PAA-pAS-SA membranes. The obtained voltages demonstrates thedifference of over 50% for the paper electrode, while the coated gold ondifferent PAA membranes did show difference of less than 15%. However,the difference from different sections of the coated gold onsame-membrane was obtained at less than 5%. When a multimeter was usedto determine the resistance of the coated gold from one end to otherend, the measured conductivity was 7.4Ω. The membrane itself was foundto be a total insulator; the multimeter reading went beyond thecalibrated level of 200 MΩ, and the instrument showed that theresistance was beyond the limit.

SEM images at 10000 and 100000 magnification of gold e-beamed PAAternary membrane and Whatman® paper are shown in FIGS. 37a and 37b .FIGS. 37a and 37b indicate that the coated gold on the PAA-SA wasuniform and even. This could be the reason of the characteristic stableresistance recorded. The obtained resistance was 15-times larger thanthat noted on the Whatman® paper, which could be related to the factthat some gold could be embedded into the PAA membrane.

Since the membranes were determined to be non-conductive according tothe 4-probe conductivity measurement, cyclic-voltammetry was furtherutilized to determine any possible electro-activity of the PAAmembranes. All of the experiments were performed in 50 mM pH 7.4 PBSbuffer. Platinum and silver wires were used as auxiliary and referenceelectrodes, respectively. Working electrodes were 200 nm gold-coated(via e-beam) Whatman® papers. Scanning rate was 50 mV, and the range was200-600 mV.

Cyclic voltammetry results of ternary PAA membranes is shown in FIGS.38a-38h . (38 a) Series 1-7 are PAA, PAA-GA (aged GA), PAA-PDA,PAA-W-GA, PAA-pAS-GA (longer drying), PAA-pAS-GA, PAA-GA (aged) longerdrying, respectively. (38 a a) overlapped of all types of PAA membranes;(38 a b) PAA, (38 a c) PAA-GA (aged GA), (38 a d) PAA-PDA, (38 a e)PAA-W-GA, (38 a f) PAA-pAS-GA (longer drying), (38 a g) PAA-pAS-GA, (38a h) PAA-GA (aged) longer drying, respectively. Ternary PAA membraneswere loaded on gold surface on e-beamed Whatman® paper. Longer dryingrefers to over 12 h drying, and while the rest were dried at than 6 h.All the tests were performed in pH 7.0 (50 mM) phosphate buffer in thepresence of silver-wire as reference electrode and platinum wire asauxiliary electrode.

TABLE M Summary of the Electrochemical Characterization of PAA MembranesPeaks for Peaks for oxidation reduction Membrane (mV) (mV) ReversibilityPAA −50 150 Quasi-reversible PAA-GA −50/100/295 130/320/470Quasi-reversible PAA-PDA 55 410 Irreversible P AA-W-GA 100 220Quasi-reversible PAA-pAS-GA −75 34 Quasi-reversible PAA-pAS-GA No peakNo peak NA PAA-GA No peak No peak NA

Conductivity of the PAA co-polymers showed dependence on aged GA and thesmall molecule type, including the time of incubation. PAA-PDA-GAprovided the highest oxidation reduction potentials (see the scale onthe y-axis for FIG. 38b ), followed by PAA-W-GA and PAA-GA. Overnightincubation under hood made PAA-GA non-conductive under the testedconditions. Similarly, PAA-pAS-GA (8 h incubation under hood) lostconductivity when it was incubated overnight under the hood. Even thoughthere was no peak for PAA-pAS-GA, recorded current seems to be 10-timeshigher than PAA. As seen from Table M, PDA and W additions masked thepeaks coming from PAA itself. PAA-PDA-GA provided the highestoxidation/reduction peaks while PAA-W-GA gave the closest distance foroxidation and reduction potentials, 110 mV. GA added two extra reductionand oxidation peaks to the membrane, and pulled PAA's reduction to 130mV from 150 mV which could be a sign that the electroactive nature ofthe aged GA and/or GA-crosslinked PAA requires less potential forreduction. GA, as shown elsewhere affects the oxidation/reduction peaksof PAA and CS, and showed the existence of additional new peak that wasnot sharp in PAA-GA and PAA-CS-GA because of that the GA was fresh andmuch diluted.

Even though PDA provides good conductivity, PDA-PMDA based PAA membranesdid not provide strong mechanical properties and durability, so GA wassubsequently introduced. PDA was first dissolved in DMAC, followed bycross-linking with GA for 30 sec; the polymerized PDA was then directlyintroduced to PAA solution. The resulting PAA-PDA-GA was casted ongold-coated Whatman® paper, followed by drying under hood for 6 h withfurther rinsing in pure water. Similarly, all of the PAA coatedelectrodes were rinsed in pure water before they were exposed to cyclicvoltammetry.

1.8 Water, Water-Vapor and Oil Permeability

FIG. 39 is a digital image of the oil-permeability test used ingathering the below data. This test was performed with the protocol of 5mL Extra virgin olive oil was put into a vial whose interior diameterand outer diameter were 20 mm and 24 mm, respectively. The testedmembranes were used to cover the open part of the vial; the vial wasplaced upside down on top of a Whatman® paper [Sigma-Aldrich, MO] asshown in the digital image in FIG. 39. The membranes were incubated for14 days at 37° C. with 95% humidity. The experiment was discontinuedafter 14 days.

FIG. 40 is a digital image of the water-vapor permeability test used ingathering the data below. This test was performed by the followingmethod; measuring the weight changes of the vial containing 5 g drydesiccant. The tested membranes were used to cover the vial entrance,and the vial was incubated at 37° C. incubator, 5% CO₂ and 95% humidity.Incubation was conducted for 7 days. In addition to this, waterpermeability of the membrane was tested with Millipore Lab-scale TFFSystem 115V (Millipore, Billerica Mass.); the pressure was kept under 30psi, and eluent-side was observed for wetness testing.

Resistance to oil and water-vapor penetration are important for keepinga containment fresh and for preventing the loss of taste and flavor.Different approaches have been applied to provide resistant surface tooil and water vapor transfer.

Due to the good mechanical properties, FIG. 1b membranes were used inthese tests. PAA-A-GA and PAA-GA were used for both tests. It was proventhat oil does not pass through the membranes. The tests were carried outin buffers and strong polar and apolar solvents including PBS buffer,DMF and hexane.

The water vapor permeability takes thickness as a parameter to determinethe power of membrane against vapor permeability by which both thequality of membrane and the importance of thickness can be evaluatedmore objectively.

Certain membranes can be used to cover the top of the vial withoutrequiring an adhesives for which only thin string or parafilm is enough.However, in some cases such as sulfanilic acid enhanced or 2BBco-polymerized PAA membranes are not easily attached with a thin stringor parafilm since they are harder plastic.

Further, PAA-I-GA, PAA-I-pAS-GA and PAA-A-pAS-GA were tested, and notransfusion was observed. These results showed that the membranessynthesized according to FIG. 1b were better than those reported onessuch as glycerol enhanced-cellulose sulfate.

Since sulfanilic acid (SA), sulfanilamide (SN), p-aminosalicylic acid(pAS) and 5-aminosalicylic acid (5AS) were used as the main molecules inthe developed membranes, PAA-pAS-5AS-SA-GA and PAA-pAS-5AS-SN membraneswere tested as well. Membranes at different thickness were tested.Thickness and texture did not affect the membranes resistant tooil-permeability.

1.9 Biodegradability and Toxicity Characterization

The synthesized membranes were heavily cross-linked with glutaraldehyde,and co-polymerized with intrinsic antimicrobial agents (i.e. sulfanilicacid, p-aminosalicylic acid). The biodegradability of these films isdetermined below. However, the introduction of these antimicrobialagents made the resulting membranes to be biodegradable. Therefore, itwas required to test biodegradability of the PAA membranes.

For the below data microorganisms were obtained from rotten sticks fromAmerican Elm (Ulmus americana) in the University's garden,Binghamton-NY. ¹H NMR, ¹H-correlation spectroscopy (COSY), and¹H-¹³C-heteronuclear single quantum coherence (HSQC). The fungus chunkwas directly introduced to the bioreactor without any selection.Pre-selected fungus and fungus chunk were characterized with molecularbiological techniques. The plasticized PAA membranes showed dramaticdifferences in terms of physical characteristics than the membranesdesigned elsewhere, so it required further biodegradation testing of thenew membranes.

Characterization of the isolated fungi species were done in theDepartment of Sustainable Bioproducts, College of Forest Resources,Mississippi State University, Starkville, Miss., USA.

FIG. 1a /1 b membranes were used in biodegradation studies because ifthese membranes can be degraded, the rest of PAA membranes synthesizedin this study can be degraded as they have lower degree ofcross-linking. PAA-GA and PAA-CS-GA have been shown to degrade in lessthan two months by the ascomycete fungus. Fusarium oxysporum.

The bioreactor contained 25 mg/mL YPD medium, 0.1 mg/mL D-glucose, 1%L-glutamine, 25 μL/mL trace-metal solution and 5 mg/mL Peptone, whichonly contains the fungi chunk that was later identified as Trichaptumbiforme. The pH of the medium was adjusted to pH 5.7 before autoclave.The bioreactor volume was 100 mL, and the membranes were from 50 mgPAA-A-GA of FIG. 1 a i and 50 mg FIG. 1 a ii PAA-GA. It also includes 50mg PAA-A-pAS-GA and 50 mg PAA-SA-GA of FIG. 1 b.

The membranes were not crushed, and put into the medium as they were.The blank bioreactor was cultured under same conditions without themembranes. Disintegration was monitored by visual decreases in themembrane size while structural degradation was monitored via NMR.Disintegration was not able to be monitored when microbial biomasstotally covered the membrane surface. In this experimental design, therewere four main differences from previous designs:

-   -   The starting fungus inoculum was prepared by dissecting the        fungi chunk, and taken the inner part as the starting biomass,        which was later on characterized as Trichaptum biforme.    -   Cells were not acclimatized before they were used to degrade the        films of FIGS. 1a and 1 b.    -   Trace metal solution was used at 25 μL/mL to enhance overall        activation of laccases and manganese peroxidases to advance PAA        degradation    -   Fed-batch process was used instead of combination of fed-batch        and continuous process

For each NMR run, 1 mL of solution from the bioreactors was put into 1.5mL polypropylene microcentrifuge tube. The sample was kept in −20° C.overnight, and then lyophilized for 24 h. The resulted solid sample wasdissolved in 0.9 mL D₂O. The dissolved sample was then left forprecipitation of non-dissolved sample for 15 min; the final volume wasbetween 0.75-0.8 mL. Degradation of the membranes was monitored via ¹HNMR, ¹H COSY and ¹H ¹³C HSQC NMR techniques.

“The trace metals solution” contained 20 mM FeSO₄7H₂0, 2 mM CuSO₄5H₂0, 5mM ZnCl₂, 20 mM MnSO₄ H₂0, 6 μM CoCl₂ 6H₂0, 1 mM NiCl₂6H₂0, and 1 mMMoCl₃.

Both Trichaptum biforme, a white rot fungus, belonging to Basidiomycotadivision of Fungi kingdom and Trichaptum biforme, similar to Fusariumoxysporum can degrade aliphatic and cyclic organic pollutants were used.Basidiomycetes are among the higher fungi that can develop multicellularmycelium. They are mainly found in rotten trees where they degradelingo-cellulosic polymers via extracellular enzymes including manganeseperoxidases, laccase peroxidase and so on.

Full ¹H spectra of the PAA for peptone-yeast medium (FIG. 41a ), at day20^(th) (FIG. 41b ), 30^(th) (FIG. 41c ), 35^(th) (FIG. 41d ), 40^(th)(FIG. 41e ) and 50^(th) (FIG. 41f ), and (FIG. 41g ) 120^(th).Degradation of the plasticized PAA membranes were monitored by examiningthe aromatic peaks belonging to PAA. The three peaks between 2-3 ppm[2.13, 2.53 and 3.10 ppm] were observed which could be thought to belongto DMAC. However, as seen in FIG. 41j , DMAC gave three peaks in ¹H ¹³CHSQC; at 120^(th) day ¹H ¹³C HSQC, these peaks were confirmed as alsobelonging to DMAC. This could be related to that addition of high amountof PAA membranes resulted in partial dissolution of the polymers whichwere surrounded by the metabolites or peptides/proteins released intothe medium. This could then prevent the release of DMAC into the medium.

DMAC is a volatile organic-solvent, and is supposed to evaporate fromthe system upon overnight lyophilization. NMR sample was prepared viafreeze- and thawing procedure where overnight-lyophilization was appliedto eliminate all the solvents coming from the biodegradation media.Disintegration of the membranes was completed within 20 days. That waswhy the 20^(th) day was selected as the first day of sampling. Aromaticregions in I H NMR spectra at 20^(th) and 30 days showed strongsimilarities; the doublets and triplets are quite similar. Aromaticregions in ¹H NMR spectra at 35^(th) and 40^(th) days showed largelytriplets in contrast to 20^(th) and 30^(th) days; 35^(th) day stillshowed some doublets and singlets which signifies the presence of PAA.However, the aromatic regions in ¹H NMR spectrum of 40^(th) day did notshow any clear evidence of the presence of PAA. Interestingly, thearomatic regions in ¹H NMR spectra at 20^(th) and 40^(th) days showedstrong similarities for the presence of triplets. For the 20^(th) and30^(th) days, a doublet was seen at 5.82 ppm which could be a sign offragmentation of the PAA molecule, which was then consumed because at35^(th) and later-days the peak disappeared.

TABLE N Characteristic peaks related to PAA degradation Day Aliphaticrange, incubation Aromatic range, ppm ppm 20 Doublets of PAA, 6.9-8.1 30Doublets of PAA, 6.9-8.1 35 Triplets, no sign of PAA 40 Triplets, nosign of PAA 50 Doublets of PAA (6.9-8.1), and triplets fromdegradation/metabolites 120 Overwhelmingly doublets Peaks of DMAC ofPAA, 6.9-8.1 (1.95, 2.7 and 3.2)

Since PAA is composed of aromatic groups, it is not expected to show anytriplet. The triplets at aromatic region are a strong sign of partial ortotal saturation of the rings found in PAA. Then 50 mg of PAA-pAS-GA andPAA-SA-GA were added to the system at 40^(th) day, which was thenanalyzed at 50^(th) day. During this period, only 0.2 mg/mL sugar wasadded to the medium at 40^(th) day. Then, the system was run incontinuous mode. From 6.5 to 8.5 ppm range of 50^(th) and 120^(th) daywas overlapped to see the changes in PAA degradation. Analyzing the50^(th) day data we found that the aromatic region in ¹H NMR revealedthe peaks of PAA and the triplets which are the signs of saturation ofaromatic groups in PAA and/or the newly formed cyclic groups. Then, 100mg of PAA-pAS-GA were added to the medium, and the mixture was incubatedfor additional 70 without adding any sugar or peptone to the system.Then, at 120^(th) day, ¹H NMR of the bioreactor was carried out; butonly the PAA related peaks were found.

When the ¹H NMR spectra were compared the followings were observed;Peptone yeast ¹H NMR possesses doublets and triplets between 3.27-3.94ppm, which is not found in any other; a singlet at 3.10 ppm, which isslightly larger at 20th and 30th days, while very small at 35th and 50thdays; a singlet at 3.30 ppm is relatively larger at 20^(th), 30^(th) and35^(th) days while it is smaller at 40^(th) and 50^(th) days; the peakis not clear for peptone-yeast medium; a singlet at 3.93 ppm probablyfound in all of them but not found in peptone-yeast medium; threesinglets between 2.75-2.78 ppm in all, but not in peptone yeast; thetriplet at 2.43 ppm found in all; a singlet 2.12 found in 20^(th) and30^(th) days, might be in 50 day; but it was not found in peptone-yeastmedium, 35^(th) and 40^(th) days; a singlet at 2.01 ppm is found in20th, 30th, 35th and 50th days, but not in peptone yeast and 40th days,this peak is relatively bigger for 20th and 30th days; a peak at 1.95ppm for 20^(th), 30^(th), 35^(th) days which is very small for 50th daywhile it was not found in 40^(th) day and peptone-yeast medium; asinglet found in all, except 30th day at 8.49 ppm; the singlets found at8.37 and 8.29 ppm only for peptone-yeast medium; the singlet at 7.84 isonly for peptone-yeast and 40th days; they are quite similar; the twosinglets at 7.6 and 7.7 are only for peptone-yeast medium; there is atriplet at 7.46 ppm found in peptone yeast medium, 20^(th), 35^(th) and40^(th) days; slight presence of the peak is in 50^(th) day while it wasnot in 30th day; the singlet at 7.10 ppm is only for peptone yeastmedium; the doublet (for 35^(th) and 40^(th) days) or the broad peak(for 20^(th), 30^(th) and 50^(th) days) at 7.22 ppm where peptone yeastmedium has a triplet. However, the doublet is relatively bigger for30^(th) day. There are 4 broad (or doublet) and a one singlet on upperfield of this peak. These are the sign of presence of PAA. However, itis not easy to say that they are present in 30th and 35th days; thebroad peak at 7.61 ppm for 20^(th), 30^(th) and 50^(th) days, which isnot included in 35^(th) and 40^(th) days; the singlet peaks between 6.5to 8.5 ppm shifted during from 20 to 50^(th) days, could be related tothe biodegradation of PAA; the triplet at 7.56 ppm for 20^(th), 30^(th)and 35^(th) days where 40^(th) day has nothing; peptone yeast medium hasa doublet and 50^(th) day has a singlet. This shows there is adegradation of PAA, but at 50^(th) day intact PAA polymers or PAApolymers protected their back-bone structure are present in the media;the two triplets and a doublet between 7.3-7.5 ppm found in 20^(th),30^(th), 35^(th) and 40^(th) day look similar, but that is not possibleto say they are from peptone or sugar since the peaks between 2-4 ppmshowed no similarity. Actually, the shapes of these triplets don't lookalike; when 50^(th) and 120^(th) days compared, there is no improvementrather the triplet at 7.46 and the doublets 7.41 and 7.35 went away for120^(th) day, and the singlet at 8.49 ppm went away as well.

Biodegradation of the membranes (PAA-CS-GA and PAA-GA) showedsome-differences such as PAA was fragmented into little fragment whichwas seen as that integrals of the four-peaks between 6.9 to 7.2 ppmbecame similar with time. For example, at 15^(th) day, the major peakwas overwhelmingly larger while at 30^(th) day they were all the same.Also, at 30^(th) day, the triplets appeared; the sign of saturation ofthe double bonds in PAA and/or conversion of the groups. However, for15^(th) to 27^(th) days, the triplets were not seen or could be verylow. Therefore, it can be said that the fungi degraded the PAA-CS-GA andPAA-GA polymers into two steps; first degraded the larger PAA polymersinto small PAA polymers. In the second step, the fungi quickly degradedthe smaller-sized PAA polymers. 10287 In contrast to this, as seen fromthe FIGS. 41a-41j , consumption of the PAA membranes synthesized in thischapter were done without requiring fragmenting the larger PAA polymersinto smaller PAA polymers. These results are summarized in Table Nabove, as a function of time of degradation. As seen from Table N, atthe 35^(th) day, all the PAA was consumed by the fungi.

Characterization of the Fungi Responsible for PAA Degradation UsingMolecular Biological Techniques

Genomic DNA Isolation-Genomic DNA was isolated from dried mycelium byuse of the NucleoSpin® Plant II Kit (MACHEREY-NAGEL GmbH & Co. KG). Thedried weight of the mycelium was 0.05 g for sample A and 0.07 g formsample B. Mycelium was washed with 95% ethanol for 2 hours. The myceliumwas transferred to tubes with 2 mm glass bead sand homogenized with CTABlysis buffer (2% cis-trimethyl ammonium boric acid, 100 mM Tris, 20 mMNa2 EDTA, 1.4 M NaCL, and 1% polyvinylpyrolidine). The extract wastreated with RNase A (200 ng/ul, incubate at 65° C. for 10 min) followedby Proteinase-K and incubated at 65° C. for 1 hour. The remainder of theextraction followed the kit instructions for isolation of DNA fromfungi. DNA was eluted with 50 μl buffer PE heated to 65° C. Theconcentration was measured on a Nanodrop 1000 spectrophotometer. Thepurity of the DNA was evaluated in gel electrophoresis on a 1% agarosegel in 1×SBA (Sodium Boric Acid).

PCR Amplification

The Internal transcribed spacer (ITS) region of the fungi was amplifiedusing primers ITS1-F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) (SEQ ID NO:1), which is specific for the higher fungi (Gardes et al. 1993), andITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (SEQ ID NO: 2), the universalprimer. Amplifications were performed in Eppendorf Mastercycler® withthe following settings: an initial hot start at 98° C. for 2 min (DNAtemplate only), melting at 95° C. for 45 s, annealing at 52° C. for 45s, and extension at 72° C. for 2 min, and final extension at 72° C. for10 min for 35 cycles. After the initial hot start, a master mixcontaining 10 mM reaction buffer, 25 mM MgCl₂, 10 mM Forward and 10 mMReverse primers, 10 mM deoxynucleotide triphosphates (dNTPs), 10 mg/mlBovine Serum Albumin (BSA), between 100-200 ng/μl total DNA isolatedfrom samples, deionized water, and 2.5 U/μl Tag polymerase was added toeach sample.

PCR products were separated by electrophoresis in 1% (wt/vol) agarosegels in 1×SBA buffer (Sodium Boric Acid) with RedGel (100 ng/ml) andrunning buffer; DNA bands were visualized by the fluorescence of theintercalated RedGel under UV light and photographed.

Sequence Analysis

The amplified fragments were inserted into the pGEM-T easy vector(Promega, Madison, Wis.) for sequencing, and the sequence of ITS regionswere confirmed by sequencing at least three individual recombinantcolonies using a Beckman Coulter (Brea, Calif.) CEQ8000 DNA sequencer.The sequence data were assembled and analyzed by the use of CEQsequencing analysis software and MegAlign (Lasergene®) and were thensearched by using the ITS-1F and ITS4 primer sequences to define the ITSregion. Each sequence was analyzed into the ITS region and was thenseparately used to perform the individual nucleotide-nucleotide searcheswith the BLASTn algorithm at the NCBI website. The outputs from theBLAST searches were sorted on the basis of the maximum identity and wererecorded. Sequence-based identities with a cutoff of 99% or greater wereconsidered significant in this study, and the best hit was defined asthe sequence with the highest maximum identity to the query sequence.

TABLE O The consensus sequence of sample Aand sample B.Consensus sequences Sample AGTTGGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAA ACGAGGGCTTTACTACTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGAGGACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAACAAGTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCTGGTTCACCAACGGAGACCT (SEQ ID NO: 3) SampleCCCGGGGCAAGGGGCGGGCGGCGTTGGATTTTGCGGGACC BCTTAACACCCGCTTCCAGCCGCGCGGGCGCCGCCGCCCCGAGGCCCGGCGCCGATCTAACAAGTAATACATCTCAAAGGTGTCCAACCGTATCCAACCAGTGGACGTCCGAGGGTCGCGCCGTTTGAGTGTCATGTTAATATCAACTCTGATGGTTTTTTGTTAATCATTGGATGTTGGACTTGGGGATCCCGTCACAGTCGACTACTGATGAGTACTATAGACTACGCATCGCGCAGCTGATATATTTAATGTCTACGTATATCAATCCATTAATAAA (SEQ ID NO: 4)

FIG. 42 shows pictures of a macroscopic and four microscopic pictures ofTrichaptum biforme (Picture of 1000× Oil Immersion).

FIG. 43 is microscopic pictures of Fusarium oxysporum (Picture of 1000×Oil Immersion).

Raw sequence data for the samples are listed below:

Sample A-1 Forward (SEQ ID NO: 5)CCGCGNGGAGGTTTCTGGACCGCTGTCCGACCGCGCCGCTCCGTTCGGCGCCGAGTTCCACTTTGTCCCCTCATTNATATTGTCAATTACGCGGGTATTCCACCGATTCCAGCTCACTTCGAAGTTGGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAACGAGGGTTTTACTACTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGAGGAACGCGAATTAACGCGAGTCCCAACACCAAGCTGTGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAACAAGTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCTGGTTCACCAACGGAGACCTGTNACAACTTTNACTCCCTCTAATGACAAAATCACTANTGAATCCCGCCGCCGCAGTCACATATGGGAGAGCTCCCACGCGTGGATCTANCTGAGTATCTATANGTCACCTAATACTGGCGTATCTGGTATACCGTCCCGGTAATGTTATCCCCCATTCCCCACTCACCGAACTAATGTAACGGGTCA  Sample A-1 Reverse (SEQ ID NO: 6)CCCTCTTTNAAATTCTTTTTAGGGGGGGGCGACTTCCCGGCGGGGCTACTCAGTCATGGATCTCTGGATGCAATAANATATTAGCGATCTTCGCCNGTGAACCACGAGGAGGATCACNAGTGCAACCCCAAACCCCTGTGAACATCCACTTGTTGCCGCGCCGATNCGNCCGCCCCCGTAAAACGGGACGGCCCGCCAGAGGACCCCTAAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCACAGCTTGGTGTTGGGACTCGCGTNAATTCGCGTNCCCTCAAATTGATTGGCGGTCACGTCAAGCTTCCATAGCGTAATAGTAAAAACCCTCGTTACTGGTAATCTCCGGCCACGCCGTAACCCCACTTTGAATGTGACCCGATCGGTAGGATACCGCGAACTAACTATATACGAG A  Sample A-2 Forward(SEQ ID NO: 7) CCGGGCGGGAGGTTTNGTTAGGGATCCCGTCGCTCGACGCGCGCCGCGCCGGTCGGCGCGCGAGTGGCCATCGGTGTCCGCCTCATTCAGTATNGTCAAGTGTGACGCGGGTATTCCTCACCCGATTCCAGGTGCACTTCCAGAAGTTGGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAACGAGGGCTTTACTACTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGAGGAACGCGAATTAACGCGAGTCCCAACACCGAGCTGTGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAACAAGTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCTGGTTCACCAACGGAGACCTTGTTACGACTTTTACTTCCTCTAAATGACCAAGAATCACTAGTGAATTCGCGGCCGCCTGCAGGTCAACATATGGAGAGCTCCACCCGTGGATGCATANCTGAGTATCTATAGTGTCCCTAATACTTGGCGTATCATGGCATACCGGTTCCGTGTGAAATGTTATCGCTCACCATCCAACAAATACNACCCGAAACTTAANGTTAACCGGGGGTCCTAATAGTGACCA CCCATTANTGCNTTGCCSample A-2 Reverse (SEQ ID NO: 8)CGGAGGTTTTTTGGGNCNCCGTCGCGACNAGGGCCCTCACTTGGAGCTCCGACCGGNCGCGCCAATTAACTCATGGATTTCGGGGATTTAGAGGAAGTAAAAAGTTTTAACAGGTGTCCCGTTGGTGAACCAGCGGAGGGATCTTACCGAGTTTACACTCCCAAACCCCTGTGAACATACCACTTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACCCCTAAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCACAGCTCGGTGTTGGGACTCGCGTTAATTCGCGTTCCTCAAATTGATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAGTAAAGCCCTCGTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAATCGAATTCCGCGGGCGCCATGGCGGCCGGAACATCAACTTCGGCCAATCCCCTATATATGTATACATCCTGGCGNTTNACAACTGGACGGGAAACGCGTACCACTATCCTGCNCATCCCTTCCCCGGCTATTCAAGCCCCCACC CTCCAATGCCCCAATGGSample A-3 Forward (SEQ ID NO: 9)CCGGAGGTNAGNCAGCACCCGCCCCTNGGAACCCNCCCATATTCTACCTGTNACCCATTTAGGCATACAATTGGGTGAACGCTGGCCCACATACCTAACAGGGCTACACTACCATGGAAGCCACTGACCGCCATCATTTGAGGAACGCAATTAACGCGAGTCCCAACACCGAGCTGTGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATTCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTACTCAGAAGTTACATATAGAAACAGAGTTTTAGGGGTCCTCTGGCGGGCCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCNAGCAACAAGTGGTATGTTACAGGGGTTGGGAGTTGTAACCGTAAT Sample A-3 Reverse(SEQ ID NO: 10) GGGCGTTATATCTTGTGGTCTCCCGCGCTTGAGGAGCTCTCCCATATGTGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTCTTGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCCTGTGAACATACCACTTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACCCCTAAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCACAGCTCGGTGTTGGGACTCGCGTTAATTCGCGTTCCTCAAATTGATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAGTAAAGCCCTCGTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAATCGAATTCCGCCGGCCGCCATGGCGGCCGGGAGCATGCGAAGTCGGGCCCAATTCGCCCTATAGTGAGTTTTATTACAATTCACTGGCCCGTCTTTTACAAACNTTGTGACTGGG Sample B-1 Forward (SEQ ID NO: 11)GGATCGCGCCGGGGGTGGGGCGGGGCCTTAAGATTTTACGAGAATTAGGTTAGAGATTTTGTCTTAGATCGAGACAGACTCAAGAATAGTTCATGGTCAAGAGTAGGATCTAACAAGTAATACATCTCAAAGGTGTCCAACCGTATCCAACCAGTGGACGGATCTTNACCGAGTTGGTGCGCAGGGGGCGCATCCCCTTGTCGAACCCACTACCCCTGGATGGCTCGTAGCTCCATCGGACGGGTGCCGGGGGGGGATCGCGTCACTGTCGANTACTGATGNGAACTATAGACTATNGATCCGGGCAGCTGATATATCCNANATCTATGTATATNAATCCATNAATAAA Sample B-1 Reverse(SEQ ID NO: 12) NNNNTGTTTTTCGGGCGCGTCGCGCGGGGCCCTCTCTGGGGAGCGTCCGCCGGNCGTCCGCCGNTTACACTAAGATGNATTTGCGAGCACGNGCTAACATGAGATAGTTATAGGCGTTNCGAGTCTTTCTACGNGAGCTCAAATCCCCTAGNTCACTGAGNCTCCCCAGCACGNGCTACAGNCCTCCTTGCAGAGAGGGGCGCTCTCTTTCGGGATCAGAATATNTACACGGGCGAAAAAAGAGGGCCCCCNTNATANCNANACNCGAGACAGTGCGACAGNCTGGACNCNGNTACACAGGTTCTGAGAGTCGNTGGNGNGGAAGAGAGTGAGACGGGNCAAACAGGGAAAACCANANAGNTCGAGTTTGTNCNGCNGTGGTNCNCNATNGGAAAAANCTCATCCCGTNGAAGGGCCCACCGANGAGCCCCCNACNAAAATNCTNGGGGTTGGGCCCGGCNCTNGTTCCNACCAAAAANGTNATNGTTCTNCTTGTAATNTCTGGGGGGGGNGTGCCCGCCCCCCNGTNCANGAATTNTANCANTANGANCGNAANAGNNTGNTGGGCAAAAACGGAGGTTCCCTCNACNCTNGAATATTAACATATTTCCCCCCCCACCAAAATATTGGTTCCTCCCACCCCGCCCCCCTTTTTGTGGGGCCCCCGCGGGTTTGGGGTTTCCAATTCCCTCGGCCTNTNTTGGCCAGAAGGAAGGTGGGGGGCNGCNGANGAAAAAAANTCCGCAAANANGGCCANGTNCAAGTTGCNACNGCNAATNGTGGGGCCTNATTTTTGGA AACCANCAATTGGGGTSample B-2 Forward (SEQ ID NO: 13)CCGGGGCNGCCGGGGCGCGTCGCCGGCNGNCNGCGGCNCNTNGGCNGCCCGCNGCGCGAGCGCAGCGNGCCGGTGGTGCNCGCGCNCACCTCCCGTCCCACCTCCTTCGCGCTCGNTGCGCNCANCTCTATANTANGTNAGAGNAGATNGAATACTAGNACTATACNTATACNTATAGCACGTAGGACGANGNAAGNGANTCNCGANATTTTTATTTGGCCGATTNTCCTATANTGNANANGGGGA AAANGGNAGNAATTTTTGAASample B-2 Reverse (SEQ ID NO: 14)CGGCGGTGGGTTTGGCTCGTGGGCCNCCCGTGCGCGGGGGGCGCCGCCTCCCTTTTTGCGGACGCGTCCNGCCCGGGCGCGCCCGNCGCGGTANACGGCTANAGTGGAGTGTGTTGCAGTGCACGNGCTATACATGGTAGTAGTTATAGGCAGTTGGGCNTGAGTACTGCTCTGTACNGGGAGNCTCAAATCCCATGAGTCCCGTGGAGGCTCCCCGACACGGGCGTACAGGCCCTCCTTTGAGAGAGGGGGCGCTCTCTTTTCCGGACAGANATATACGCGGGCGAAAANGAGGGCCCNCNTTTNTNTCGGNACNCNAGGGTCANGTNCNGAGCANGNTCNTAGNACCCCCCGGGGAAACAACANGGTTTTNCTCGACGAAAGTNCGNGTGGGGGCGGGGGGAAAGAACCAAGTNGAAAGAACGGGGGCCCANTAACAGGAGGAAAAACCCAAAGANGANTCNGAATTTGTNCCNCNGTGGTNAACCNATNGGAANGANCTTATNCNGTNGAAGGGCCNAGNGANGAGCCCCCNACNGACATNCTTGGGGGTTGGGCCCGGCNCTNGTTCCCAACCAANACCGGTTAATNGTTCCTCCCTTGTTTAATNTCTGGGGGGGGGGTNNGTGCCCCGGCCCCCCCCTCGGTTCAAAAGAAATTTNTAACCAAANAAGGAACGCAAAAAAGNNTGNGTGCCAAAAACCGNAGGTTCCCTCNACNCTNGAATTANACNNATTCCCNCGCCACCAAANATTTGTTCCTCAACNCGGCCCCCTTTTGTGGGGCCCCCGGGGTTTGG TGTTTCTAAATTCCTTGGCSample B-3 Forward (SEQ ID NO: 15)CCCGGCGAATGTTTTATGGGGTCATGTTCGACCGCGCCGTCCGGTTGGCGGAGTTNCATTTTCGTGATCTANAAGAGATAAAATGGCTAAACAGGTTTACCGTAGGTTATTANCCGCGGAAGGATCTTAACAGTTTTGAAGTGGGCTTGATGCTGGCTTGTAACAGAGCACTGTGCTCAGTCCCGCTCCAATCCATTCAACCCCTGTGCACTATTCGGAGTGTTGCAAGCTAAGACAATGTGGGGAGTGGTCCCGGTTGTATTTCTAATGCGACTTGGGCTTACTTTCAAACGGTCAAGGCTTGTCCTCCGGTTTATATACAAACACTTTTATTGTCTTGTCGAATGTATTAGCCTCTCGTTAGGCGAAATTTAAATACAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGCTATTCCGAGGAGCATGCCTGUTGAGTGTCATGTTAATATCAACTCTGATGGTTTTTTGTTAATCATTGGATGTTGGACTTGGAGGTTCGTGCTGGCTGCAAAGTCGGCTCCTCTTGAATGCATTAGCTTGGACCTGTGCGCGTTTGCTAGCGGTGTAATACATTTAATTCACCACGGGCCGTGTCACTATTAGGGTCTGCTTCTATTCGTCCTACCGGACAATAATAACTTATGACCTGACTCAATAGGTAGACACCCCGACTAACTTAATACCGAGAATCANTATCCGCCCGCGTACATGAAA Sample B-3 Reverse(SEQ ID NO: 16) CCAGAAGGATTTNATGAAACAAGATAAGCAGAGGTCCCTCATCTTNGGACTCCGACGGCGNCGCCATATAACTCATGATTTCCCGCTCTATTGATATGCTAAGTTTTTAGCGGGTAGTCCACCGATTTGAGGTCAGAGTCATAAAGTTTATTATTGTCCGGTAAGGACGATTAGAAGCAGACCCTAATAGTGACACGGCCCGTGGTGAATAAAATGTATTACACCGCTAGCAAACGCGCACAGGTCCAAGCTAATGCATTCAAGAGGAGCCGACTTTGCAGCCAGCACGAACCTCCAAGTCCAACATCCAATGATTAACAAAAACCATCAGAGTTGATATTAACATGACACTCAAACAGGCATGCTCCTCGGAATAGCCAAGGAGCGCAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGTTGAAAGTTGTATTTAAATTTCGCCTAACGAGAGGCTAATACATTCGACAAGACAATAAAAGTGTTTGTATATAAACCGGAGGACAAGCCTTGACCGTTTGAAAGTAAGCCCAAGTCGCATTAAAAATACAACCGGGACCACTCCCCACATTGTCTTAGCTTGCAACACTCCGAATAGTGCACAGGGGTTGAATGATGGAACGGACTGACACAGTGCTCTGTACAGCCACATAAGCCACTCAACTCGTATGATCTTCCGCAGTACTACGAACTGTACATTTATTCCCTATACA

The Sample B fungus used in this study was taken from a rotted-elm treein Binghamton University Garden, Binghamton-New York. Sample A funguswas isolated semi-selectively from sample B which was grown on NutrientBroth Medium, Trametes defined medium, and Candida albicans selectivemedium. The candidate fungi A and B were submitted for molecularcharacterization to the Molecular Biology lab in the Department ofSustainable Bioproducts at Mississippi State University.

TABLE P Comparison of Gen-Bank top hits for the ITS region No. of ITSmatches/ Organism ITS % ITS no. identified Isolate identified identityin GenBank Sample A Fusarium 100% 328/328 oxysporum Sample B Trichaptum 99% 551/558 biforme

The characterization results showed that the fungi selection in theprevious study lead selection of Fusarium oxysporum over Trichaptumbiforme; it should be noted the fungi chunk can have some impurities.

Polycyclic organics can be degraded by Fusarium oxysporum and Trichaptumbiforme, as well as, Penicillium italicum (P. italicum), Glomerellacingulata (G. cingulata), Aspergillus flavus, Colletotrichum alatae,Fusarium solani, Ceriporiopsis carnegieae, and Xenoacremonium falcatus.The type of extracellular enzymes released to the medium by these fungi,and their growth pattern and performance have an impact on their PAAdegradation, which might be the reason for the time required to achievefull degradation of the membranes.

Full degradation refers to when the fungi have completely consumed thePAA membrane. As seen from the Table N above, 1H NMR did not show anypeak related to PAA polymers and the polymer has been totally, or nearlytotally, degraded.

Cytotoxicity Characterization of Ternary PAA Membranes

PAA membranes did not show any cytotoxic effects on non-cancerous andcancerous cell lines. Since the membranes synthesized here aredifferent, their cytotoxicity on non-cancerous IEC6 and cancerous A549cell lines were tested as well. Two different membranes from FIG. 1a and4 membranes from FIG. 1b were used.

FIG. 44a is an illustration of membrane loading in a well. As seen fromthe graphics in FIG. 44b , PAA-GA of FIG. 1a , and PAA-A-pAS-GA,PAA-I-pAS of FIG. 1b did not show any significant cytotoxicity, whilePAA-5AS-GA of FIG. 1b showed significant cytotoxicity, for whichviability decreased at nearly 40%. However, it should be mentioned thatlow concentration of 5AS (below 0.3 mg/mL) was not toxic to the cells.

PAA-A-GA of FIG. 1a and PAA-W-GA of FIG. 1b membranes showed unexpectedresults; cells did not only grow in the wells, but also they grew on themembranes. SEM images of the membranes are seen below. The surfaces wereeither woven-like or micro-porous, which can allow cells to grow onthem. 3D cell culture provides unique environment for seeded cells torecapitulate in vivo conditions. Biocompatibility, porosity andhydrophilicity are important for 3D cell culture materials. High-masstransport, ease of process, flexibility and good-mechanical strength isthe desired properties of 3D cell culture material, which limit choiceof 3D cell culturing materials.

Good 3D cell culturing supports possess high transparency andlow-background fluorescence ability for high quality light microscopyand fluorescence imaging. Stiffness of the support material is a factorfor proper cell migration because cells must apply cytoskelatal forcesfor movement instead of passive-movement driven by fluidity of thesupport material or the system. This is possible by providing solidstiff support materials.

Antimicrobial Activity of the Films

The featureless membranes synthesized according to FIG. 1b were utilizedcharacterized for antimicrobial studies. Staphylococcus epidermidisATCC® 12228™, Escherichia coli ATCC® 25922™ and Citrobacter frenduiiATCC® 8090 were cultured in Mueller-Hinton broth at room temperature for24 hrs. The viable cell number was determined by conventional agarplate. The resulting figures were Detailed explanations were given underrelated figures.

Antibacterial activity of ternary PAA membranes. FIG. 45a 0.2 mg/mL Ilewas dissolved in GA and then added to 0.16 M PAA. Overall GAconcentration was % 0.1. FIG. 45b 3 mg/mL 5AS was dissolved in 0.16 MGA, and then % 0.2 GA was added to the system. Both membranes wereincubated at RT for 12, and then membranes were peeled off. 10⁴ cfu/mLfor one area and 10⁶ cfu/mL for the three sections were inoculated forboth membranes. While Ile did not show any antibacterial activity, 5ASdid not allow bacterial development. Incubation was 72h. FIG. 45c 0.2mg/mL PCAM sugar was dissolved in 0.16 M PAA, followed by 0.2 mg/mL Ilewas dissolved in GA and then added to 0.16 M PAA-PCAM. Overall GAconcentration was % 0.2. FIG. 45d 3 mg/mL PAS was dissolved in 0.16 MGA, and followed by W addition [1 mg/mL W dissolved GA was added to thesystem]. Overall GA concentration was % 0.2. Both membranes wereincubated at RT for 12, and then membranes were peeled off. 1500, 150,15 and 1.2 cfu/mL added to the different area. While Ile-PCAM did notshow high antibacterial activity [600 colonies formed out of 1500 cfu,and 26 cfu out of 150 cfu], PAA-W/GA-PAS did not allow bacterialdevelopment. Incubation was 24 h for FIGS. 45c and 72h for FIG. 45 d.

The introduction of pAS or 5AS was found to advance the antibacterialactivity of the disclosed films. Antibacterial activity refers to thefact that the disclosed film will not cause the growth of bacterial andhence will preserve contained food.

To visualize bacterial development, plate counting method was utilized.Incubations were made up to three days, and no bacterial colonies wereobserved. The disclosed films provide about a 99.999% reduction ofbacterial growth.

Similarly, 5AS, pAS and pAB enhanced PAA membranes showed goodantibacterial activity against Aeromonas hydrophila, Pseudomonasaeruginosa, Escherichia coli DH5alfa, Listeria monocytogenes strainsF2365 and HCC7. Good antibacterial activity of the PAA membranes canalso be displayed against other gram-positive and/or gram-negativebacterial species. Gram-positive species other than Listeriamonocytogenes, can include Staphylococcus epidermidis. Gram-negativespecies other than Escherichia coli, Aeromonas hydrophila, can includeEnterobacter aerogenes and Citrobacter freundii.

Virulent type strain L. monocytogenes were grown in a rich medium suchas brain heart infusion (BHI). Lysogeny broth (LB), a nutritionally richmedium agar also used for the maintenance of the tested E. coli. Thebacteria were taken from the culture collection unit −80° C. freezer indepartment of Basic Science, College of Veterinary Medicine, MississippiState University Mississippi-USA.

Even though 5AS enhanced-PAA membranes showed that good antimicrobialactivity includes showing antifungal activity, it disrupted membranemechanical properties with over 0.5 mg/mL usage while pAS can be used upto 2 mg/mL for PAA membrane preparation. So, the packaging membrane cancontain 0.5 mg/mL pAS and 0.1 mg/mL 5AS, which provides good physicaland antimicrobial properties. However, it should be mentioned thatselection of antibacterial molecule is also affecting the color, soinstead of pAS/5AS, pAS and 5AS can be used independently at differentconcentrations.

As seen from FIGS. 45a-45k , pAB can be used instead of pAS since itshowed similar antibacterial activity. 2×10⁷ cfu of E. coli and S.epidermidis in 20 μL were dropped on the agars shown in FIGS. 45e-45hand FIGS. 45j -45 k. 0.2 mg/mL Ampicillin were dissolved in agar placedin well FIG. 45e while same amount of ampicillin was put on left side ofwell FIG. 45h ; combination of 0.1 mg/mL 5AS and 0.2 mg/mL pAS weredissolved in agar placed in well FIG. 45f while same composition was puton left side of well FIG. 45 j; combination of 0.1 mg/mL 5AS and 0.2mg/mL pAB were dissolved in agar placed in well FIG. 45 g while same thecomposition was put on left side of well FIG. 45 k.

For well FIG. 45h , slight bacterial growth was observed in comparisonto well FIG. 45j and FIG. 45k ; this could be related to that ampicillindissolved in agar while combination of 5AS and pAS or 5AS and pAB didnot dissolve. When ampicillin was dissolved in agar, no bacterialformation was observed. Similarly, combination of 5AS and pAS eliminatedall the bacteria while combination of 5AS and pAB did only wipe out99.99%. Therefore, the good antibacterial activity of 5AS/pAS and5AS/pAB modified membranes was obtained in comparison to non-antibioticcontaining PAA membranes. However, in the case of sulfanilamide basedmembranes, utilization of pAS, 5AS or pAB are not required at higherlevels since sulfanilamide membranes showed antibacterial activities.

For the well in FIG. 45l , a control of Pseudomonas aeruginosa in agaris shown, while FIG. 45m contains PAA-SA-pAS-5AS-GA and FIG. 45nPAA-SA-pAS-GA and FIG. 45o PAA-SA-pAS-W-GA for the membranes preparedaccording in DMAC. FIG. 45p PAA-pAS-5AS-GA and FIG. 45r PAA-SA-pAS-GAmembranes prepared in 60:40, Ethanol/DMAC, solvent. All of the membranesshowed strong suppressing effect (cidal effect was well) on Pseudomonasaeruginosa; synthesizing the membranes in ethanol:DMAC mixture did notshow any negative effect on membrane's duty. Here, another importantresults were observed that introduction of 5AS to the membrane enhancedits antibacterial activity which was observed as smaller colonyformation in contrast to only pAS containing membranes.

Aeromonas hydrophila was tested on membranes for 24h and 48h incubation.For FIGS. 45s-y , incubation period was 24h. FIG. 45s is control for 24incubation period; FIG. 45t PAA-SA-pAS-5AS-GA; FIG. 45u PAA-pAS-5AS-GA;FIG. 45v PAA-SA-pAS-GA (solvent 65:35, Ethanol:DMAC); FIG. 45wPAA-SA-pAS-5AS-GA (solvent 60:40, Ethanol:DMAC); FIG. 45y PAA-SA-pAS-GA(solvent 60:40, Ethanol:DMAC). For the membrane FIG. 45y , the bacteriadid not grow on membrane, but around the membrane microbial colonieswere observed. For FIG. 45z -ae, incubation period was 48 h. FIG. 45z iscontrol for 48 h incubation; FIG. 45 aa PAA-SA-pAS-5AS-GA membrane; FIG.45 ab PAA-SA-pAS-W-GA membrane: in the cases of losing membranesintegrity causing A. hydrophila grow on membrane while the protectedarea of the membrane did not allow bacterial growth. Thus, it appearsthat the antibacterial activities of the membranes were not coming fromreleases of pAS or 5AS into the media.

As a note, as seen from FIGS. 45f and g , free pAS and 5AS were wipedout both gram (+) and gram (−) bacteria. So, it can be said that GAmight cross-link pAS and 5AS, and when they were released into themedia, they do not show strong antibacterial activity while when theywere bonded to the PAA, they were more active.

FIG. 45 ac PAA-SA-pAS-5AS-GA membrane; likewise, FIG. 45 ab, when themembrane lost its integrity, the membrane allowed bacterial growth. FIG.45 ad PAA-SA-pAS-5AS-GA membrane while FIG. 45 ae PAA-SA-pAS-GAmembrane. Membranes eliminated A. hydrophila development strongly aslong as the membrane protected its structure.

FIG. 45 af is a control of Listeria monocytogenes for 24h incubation;FIG. 45 ag PAA-SA-pAS-W-GA: L. monocytogenes showed growth on somesections of the membrane where the membrane integrity got lost; FIG. 45ah PAA-SA-pAS-5AS-GA FIG. 45 aj PAA-pAS-SA-GA; FIG. 45 akPAA-pAS-5AS-GA; FIG. 45 al PAA-SA-pAS-GA (solvent 65:35, Ethanol:DMAC).

Antifungal activity of some ternary PAA membranes is shown in FIGS. 45am-45av. The fungi used here was Aspergillus nidulans. Incubation periodwas 6 days for all these membranes. FIG. 45 am PAA-SA-pAS-W-GA; FIG. 45an PAA-SA-pAS-GA; FIG. 45 ap PAA-SA-pAS-5AS-GA; FIG. 45 arPAA-pAS-5AS-GA; FIG. 45 as PAA-SA-pAS-GA; FIG. 45 at PAA-SA-pAS-5AS-GA(65:35, Ethanol:DMAC solvent); FIG. 45 au PAA-SA-pAS (60:40,Ethanol:DMAC solvent); FIG. 45 av PAA-SA-pAS-5AS-GA. For all themembranes, where there was disintegration, the fungi showed growthpattern. However, the growth was not all over the membrane. For FIG. 45ar, S. aerugenosa and A. nidulans were inoculated together; while thefungus showed growth at the edge of the membrane, the bacteria did notshow any growth. For FIG. 45 av, when there is distortion on themembrane, development of fungus was observed while the protected side ofthe membrane did not allow fungus development.

Poly(amic)acid polymer has been synthesized from 4,4-oxydianiline (ODA)and pyromellitic dianhydride (PMDA) in anhydrous N,N-dimethylacetamide.Three procedures have been applied to develop antibacterial andantifungal PAA membranes and thin films. (i) entrapping p-aminosalyclicacid (1 mg/mL) into PAA thin film, (ii) polymerizing p-aminosalyclicacid (1 mg/mL) via glutaraldehyde, followed by introduced to the PAAthin-film, and (iii) entrapping p-aminosalyclic acid (1 mg/mL) in PAAmembrane. Thin films were prepared via controlled solvent evaporationmethod while membrane was prepared via coagulation-based phase inversionwhere 2 h controlled evaporation under hood was applied to increasepore-size. 1 mg/mL p-aminosalicyclic acid was shown to eliminate both E.coli and S. epidermidis under the testing conditions.

Scanning Electron Microscope (SEM) images of FIG. 46a PAA thin film andFIG. 46b PAA membrane which contains p-aminosalyclic acid cross-linkedwith GA and p-aminosalyclic acid molecules, are shown respectively.

Antibacterial activity of p-aminosalyclic acid supported PAA thin filmsand membranes on Escherichia coli ATCC 25922 (E. coli, gram−) andStaphylococcus epidermidis ATCC 12228 (S. epidermidis, gram+) are shownin FIGS. 47a-47h . FIG. 47a is a control of E. coli and S. epidermidis;FIG. 47 b E. coli and FIG. 47 c S. epidermidis inoculated onto the PAAfilm which contains p-aminosalicylic acid was cross-linked withglutaraldehyde, followed by rinsed and dried in anhydrous methanol toquench further cross-linking; FIG. 47 d E. coli and FIG. 47e and FIG. 47f S. epidermidis inoculated onto the PAA film which entrapsp-aminosalyclic acid; FIG. 47 g E. coli and FIG. 47 h S. epidermidisinoculated onto the PAA porous membrane which entraps p-aminosalyclicacid.

The results indicate that capturing p-aminosalicylic acid in PAAmembranes did not show a descent antibacterial activity towards both E.coli and S. epidermidis. However, as seen from FIG. 47f , suppression ofbacterial development is shown; the film was thinner in comparison tothe film of FIG. 47e , which depicts that thinner film was more prone torelease its containment to show its antibacterial activity. As seen fromFIG. 47b and FIG. 47c , bacterial growth for E. coli and S. epidermidiswere suppressed, particularly the thin film didn't allow much bacteriato grow on the thin film, but still S. epidermidis grew on the membrane.In contrast to the thin films, nanostructured PAA membrane showed highantibacterial activity (i.e. up to about 90%) towards both E. coli andS. epidermidis.

The results indicate that thickness of PAA thin film and eligibility ofthe molecule to transfer from inside of the membrane to the media (PAAmembrane), and possible non-covalent modifications done on thin filmenhances their antibacterial capability.

1.10 Surface Characterization

Surface characterization of the membranes synthesized according to FIGS.1a and 1b are provided together. Description of the membranes andprocedures are given under the related figures.

SEM image of PAA membrane of FIG. 1a are shown in FIGS. 48a and 48b .FIG. 48a top and FIG. 48b bottom phases of PAA. This membranesynthesized as followed; the viscous solution from 0.18 M PAA solutionwas casted on glass and incubated in air-tight cabinet for 6h, and thenphase-inverted under hood for 12h (FIG. 1a -ii).

SEM image of PAA-A-GA membrane of FIG. 1 a are show in FIGS. 49a and 49b. FIG. 49a top and FIG. 49b bottom phases of the membrane. This membranesynthesized as followed; 0.25 mg/mL GA was added to 0.20 M PAAcontaining 1 mg/mL A, and stirred for 2 min. Then the viscous solutionwas casted on glass and incubated in air-tight cabinet for 6h, followedby 3h incubation in hood and then phase-inverted in pure water for 3h.It is clear that the localized pores or deposits possibly from L-alaninecouple to PAA via GA; this is characteristics of the membranes preparedaccording to FIG. 1a . However, when the molecules totally dissolved inPAA viscous solution of FIG. 1 a, they provided featureless surface.

SEM image of PAA-A-GA membrane of FIG. 1a -ii are shown in FIGS. 50a and50b . FIG. 50a top and FIG. 50b bottom of the membrane. This membranesynthesized as followed; 0.25 mg/mL GA was added to 0.20 M PAAcontaining 1 mg/mL A, and stirred for 2 min. Then the viscous solutionwas casted on glass and incubated in air-tight cabinet for 6h, and thenphase-inverted in anhydrous ethanol for 2h, followed by 3hphase-inversion in pure water. FIG. 1a -ii, this membrane did notrequire sonication, however sonication is possible.

SEM image of PAA-A-GA of FIG. 1b -ii are shown in FIGS. 51a and 51b .FIG. 51a is top face, and FIG. 51b is bottom face of the membrane. 2mg/mL Alanine was added to the PAA solution. 100 μL/mL GA from 25% GAwas introduced to 5 mL PAA solution. 20 μL/mL methanol was introduced tothe 0.18 M PAA solution The glass surface was then wetted with drymethanol, and then the solution was casted on the glass on whichmethanol was then spreaded, then incubated at room temperature for 6 h.Finally, the membrane was dried under hood for 12 h. Both faces have nopores. FIG. 1b -ii, the major difference of this PAA-A-GA membrane fromother PAA-A-GA membranes was that A was pre-dissolved and treated inGA-water, which resulted in elimination of localized porous-areaformations.

SEM image of PAA-CA-GA of FIG. 1b are shown in FIGS. 52a and 52b . FIG.52a is the top face and FIG. 52b is the bottom face of the membrane. 2mg/mL cellulose acetate was added to the 0.18M PAA solution. 100 μL/mLGA from 25% GA was introduced to 5 mL PAA solution. 40 μL/mL ethanol wasintroduced to the PAA solution The glass surface was then wetted withdry ethanol, and then the solution was casted on the glass on whichethanol was then spreaded, then incubated at room temperature for 6 h.Finally, the membrane was dried under hood for 12 h. Both faces have nopores. FIG. 1b -ii.

SEM image of PAA-SA-pAS-GA membrane of FIG. 1b are shown in FIGS. 53aand 53b . FIG. 53a top and FIG. 53b bottom phases of the membrane. Bothof the surfaces are featureless.

SEM images of PAA-W-GA membrane are shown in FIGS. 54a -54 d. 10 mg Trp(W) is dissolved in 100 μL GA from 25% GA stock. The Trp was incubatedfor 20 sec for polymerization with the help of GA. Then, the Trp-GAsolution was introduced to 0.16 M PAA (or 0.12 M) solution at 5 drops,and 5 second interval was followed each consecutive drops. 3 minstirring after last drop of Trp-GA, the PAA solution was casted onglass. 4 h incubation at room temperature, followed by 12 h incubationunder-hood. Finally, the membrane was sonicated in anhydrous methanolfor 20 min, which was then dried under hood. FIGS. 54a and 54b areimages were taken with inlens detector, while FIG. 54c and FIG. 54dimages were taken with SE2 detector. Sonication of the same membrane inpure water, 20% MeOH and 50% MeOH did not provide any pore-formation.However, 80% EtOH allows pore-formation.

SEM images of PAA-W-GA and PAA-SA-GA membranes are shown in FIGS.55a-55f . FIG. 55a top and FIG. 55b bottom of the PAA-SA was incubatedat room temperature for 4 h, followed by incubated in 70% Ethanol inwater for 2h. FIG. 55c top and FIG. 55d bottom of the PAA-W wasincubated at room temperature for 4h, followed by 2h incubation in 70%Methanol. FIG. 55e top and FIG. 55f bottom of the PAA-SA was incubatedat room temperature for 4h, followed by 2h incubation in 70% Methanol.PAA-SA gets solidified faster than PAA-W; so it is normal to see lessporous surface for PAA-SA. As seen from all of the FIGS. 54a-54d andFIGS. 55a-55f , the surfaces of the membrane are porous enough for celladhesion; since both sides showed pores, transfer of wastes resultedfrom cellular metabolism is possible.

Surface characteristics of all synthesized PAA membranes did not showany difference in response to alteration in PAA concentration (from 0.08M to 0.20 M range), GA concentration and small molecule and itsconcentration. However, at macro-scale all parameters affected theeye-visible membrane surface. Addition of organic solvents includemethanol, ethanol and tetrahydrofuran did not make any difference onsurface characteristics. Here, the most dramatic change in surfacecharacteristics were seen in parallel to changes in procedure. Here,three major surface types were obtained; featureless, macro-porous andwoven-like surfaces. Nearly all the procedures provided featurelessmembrane surface; pore-free surfaces can be a good barrier againstpenetration of oil, water-vapor and gas transfers.

SEM image of PAA-GA are shown in FIGS. 56a and 56b ; FIG. 56a top andFIG. 56b bottom phases of PAA-GA membrane. This membrane synthesized asfollowed; 0.25 mg/mL GA from stock was added to 0.18M PAA solution andstirred for 3 min, and then the viscous solution was casted on glass andincubated in air-tight cabinet for 12h, and then further incubated underhood for 6h. The membranes synthesized according to FIG. 1a -i.

The right figure is top face FIG. 57a , and left one FIG. 57b is bottomface of the membrane. 5 mg Alanine was dissolved in 100 μL of 25% GAsolution. The cross-linked L-Alanine was then introduced to 5 mL 0.18 MPAA solution. 40 μL/mL methanol was introduced to the system. The glasssurface was then wetted with dry DMAC, the PAA solution was casted onthe glass and then incubated at room temperature for 6 h. Finally, themembrane was dried under hood for 12 h. Bottom is nearly featurelesswhile the top has recognizable numbers of defects. FIG. 1b -ii.

As illustrated and discussed above, both FIG. 1a and FIG. 1b can providefeatureless surface if the small molecule or cross-linker are dissolvedin PAA solution properly.

1.10 PAA Concentrations

PAA concentration was selected as 0.16, 0.18, 0.20 and 0.25 M based onthe pore size of pure PAA membrane obtained via phase-inversion. PAAsolution's viscosity was not measured with an instrument; if theviscosity is low enough to be stirred with stirring magnet at highspeed, the concentration was accepted as good. The followingoptimizations were obtained from FIG. a. 0.18 M was selected as bestconcentration for FIG. 1 a subsequent membrane preparation.

In addition to viscosity, other parameters were considered including theratio of ODA to PMDA, temperature of the medium and the speed ofstirring showed great impact on PAA formation; in optimum conditions,the PAA concentration is 0.08 M in the cases of 1.00:1.03 ODA:PMDA ratioat 40° C. under mild mixing (i.e. between 400-600 rpm). The mixingshould be enough to totally dissolve PMDA at less than 120 seconds butno earlier than 30 sec. In the case of 0.12 M, ODA:PMDA a good ratio wasfound to be between 1.00:1.04 at 40° C. under mild mixing to obtainviscous PAA solution. However, the formed PAA solution was highlyviscous.

In the case of high temperature such as 70° C., PMDA was dissolved inseconds and the resulting 0.12 M PAA solution was not as viscous at1.1:1.0 ODA:PMDA ratio. Similar results were observed for 1:1 ODA:PMDAratio at high temperatures. At 40° C., 1.00:1.10 ODA:PMDA ratio, 0.12 MPAA became less viscous than 1:1 ratio. So, it can be concluded that agood PAA concentration is 0.08 M or 0.12 M for FIG. 1b membranes.

TABLE Q Summary of optimization of ODA:PMDA ratio, stirring speed andtemperature needed to produce viscous PAA solution. Observation ODA:PMDAStirring viscosity low, ratio speed Temperature Time high, same?1.00:1.00 400 25° C. Overnight Mild 1.00:1.01 400 25° C. Overnight Mild1.00:1.02 400-600 40° C. Overnight High 1.00:1,02 400 50° C. OvernightVery high 1.00:1.02 400 60° C. Overnight Mild 1.00:1.02 1200 70° C.Overnight Very low 1.00:1,02 400-600 70° C. Overnight Low 1.00:1.04 40040° C. Overnight Mild 1.00:1.05 400 40° C. Overnight Low  1.2:1.0 40025° C. Overnight Very low

The choice of appropriate solvent depends on three parameters as (i)environmental-friendliness, (ii) chemistry such as reaction yield and(iii) engineering which is more of scalability and ease of down-streamprocess. Environmental aspects of solvents are regulated by USEnvironmental Protection Agency (EPA). Chemistry and engineering aredefined by the process itself and aim of the study.

The solvents aim to meet the following requirements: the solvent must beneutral to all members of the reaction mixture, including reactants,products as well as catalysts; if the solvent is a provider of any groupsuch as —O, —H, that should be just a carrier; the solvent must beliquid at the reaction condition such as if the reaction is happening atroom temperature the solvent should not require higher degrees to beliquid; if the phase split is required for the solvent that should bepreferred; the solvent should provide the desirable solubility for thereactants as well as products if it is required; the solvent should notundergo association or dissociation; and the solvent should selectivelydissolve reactants or the possible products if it is desired.

In order to obtain “greener” membranes (those with a smaller ecologicalimpact), DMAC was combined with ethanol at varying ratios. Ethanol isgenerally accepted as a “green” solvent. However, the criteria listedabove were taken into account in the selection. The goal was to use ashigh a percentage of ethanol as possible while minimizing the volume ofDMAC employed. Ultimately, an optimum condition is sought that willprovide the highest benefit. Determining the possible highestEthanol:DMAC ratio was done using only two parameters; (i) the physicalproperties of the membrane and (ii) the aim of application of themembrane.

The observed characteristics of the membrane were (i) durability, (ii)resistance against solvents and (iii) mechanical properties. 65:35Ethanol:DMAC ratio was accepted as a good ratio to develop 0.12 M PAAmembrane with 1.00:1.03 ODA:PMDA ratio at 50° C. medium temperatureunder mild mixing. However, the PAA polymer formed in 65:35 Ethanol:DMACratio did not form fluorescent active membrane, so it is not advised forfluorescent active membrane formation. Actually, introducing ethanolinto PAA solution prepared in only DMAC still disrupting formation offluorescent active membrane formation.

TABLE R Determining good conditions for Ethanol:DMAC ratios in membranepreparation. Mixture Observation 50:50, DMAC:EtOH Viscosity high,require warming up (i.e 50° C.), and forming membranes that are strongbut hard to obtain different colors 35:65, DMAC:EtOH Viscosity mild,require warming up (i.e 50° C.), and forming membranes that are strong,but hard to obtain different colors 35:65, DMAC:EtOH Viscosity mild,require warming up (i.e 50° C.), and forming membranes are strong,require the addition of 2% water to obtain desired colors 25:75,DMAC:EtOH Viscosity acceptable, require warming up (i.e 60° C.), andforming membranes that are strong, but hard to obtain different colors35:50:15, Viscosity acceptable, require warming up (i.e DMAC:EtOt:Water60° C.), and require special care to form good membranes, but providedesired different colored membranes 30:50:20, Viscosity low, requirewarming up (i.e 60° C.), DMAC:EtOH:Water and require care to form goodmembranes, but provide desired different colored membranes 60:40,DMAC:Water Did not form PAA viscous solution 60:30:10, Did not form PAAviscous solution DMAC:Water:AcOH *PAA concentration employed was 0.12for all experimental conditions indicated.

Water:Ethanol:DMAC and Ethanol:DMAC mixtures were tested as well. In allcases, PAA concentration was kept constant at 0.12 M. The followingsolvent mixtures were obtained a good reachable ratio as 15:50:35(water:ethanol:DMAC) and 75:25 (Ethanol:DMAC). However, it was shownthat 20:50:30 (water:ethanol:DMAC) ratio is possible, but the PAAsolution should be used within 2 days, otherwise PAA precipitates in thesolution due to presence of high water content and low DMAC ratio.

However, it was noted that introduction of water to the solvent systemeliminates the ethanol effect of preventing colorful and fluorescent PAAmembrane formation. Methanol was not tested with combination of DMAC;ethanol is less toxic in comparison to methanol. However, methanol,ethanol and water containing 0.1 M hydrochloric acid (HCl) were testedindividually, but viscous PAA solutions were not obtained.

Optimization of Small Molecule Concentrations

Optimization of the small molecule concentrations were performed for themembranes synthesized according to FIG. 1 a. Since the small moleculesused were mostly insoluble in viscous PAA solution. Therefore, it was agoal to find good concentrations of the molecules in order to formuniform and stand-alone membranes. Changes in viscosity related to smallmolecule addition was not tested with an instrument, rather theviscosity was described as low, mild, high and very high in relation tomembrane preparation; and ease of spread onto the glass substrate priorto phase inversion. low and very high viscous PAA-small molecules werenot be able to cast on glass surface to form even membranes.

However, for the membranes synthesized according to FIG. 1b , the smallmolecules were pre-dissolved before being introduced to the viscous PAAsolution, hence optimization of the concentrations was performed mostlywith respect to the aspect of mechanical strength and antimicrobialactivity.

Structures of the small molecules used in FIG. 1a membranes are shown inFIGS. 58a-58m . FIG. 58a Chitosan, FIG. 58b cellulose acetate, FIG. 58cglucosamine, FIG. 58d L-alanine, FIG. 58e L-lysine, FIG. 58f L-cysteine,FIG. 58g L-isoleucine, FIG. 58h L-tryptophane methylester, FIG. 58jglycine, FIG. 58k glutamic acid, FIG. 58l L-arginine and FIG. 58mL-threonine.

For all PAA concentration, 2 mg/mL chitosan (CS) [Low-molecular weightchitosan, Sigma-Aldrich] concentration can be used. CS mediated increasein the viscosity of PAA solution showed distinct characteristics; At 0.5mg/mL, 1 mg/mL and 3 mg/mL of CS, PAA solution became highly viscous.From observations, CS is insoluble in DMAC: so it is clear thatyellowish CS flanks are in PAA solution, which causes un-uniformity.

Cellulose acetate (100 kDa molecular weight) completely dissolvedresulting in a clear solution when mixed with PAA solution despite thefact that cellulose has similar structure to CS. PAA and PAA-CAsolutions exhibit similar color and uniformity with no air bubble inPAA-CA unlike PAA-CS mixture. The formation of the air bubbles resultedfrom combination of insolubility of the small molecule and highviscosity. When the PAA-small molecule mixture was stirred at 100 rpmfor 10-30 min, the air-bubbles disappeared. Interestingly, CA at 1 mg/mLconcentration makes PAA solution highly viscous which is like solid, soit is concentration should be used less than 0.5 mg/mL. Even at 0.5mg/mL concentration, PAA-CA solution became viscous which made iteligible for membrane preparation by water-bath mediated phaseinversion.

D-glucosamine (DA) is one of the two monomers in chitosan molecule, sothey were expected to possess similar properties. Similar to CS, DA wasnot soluble in DMAC. However, when 1 mg/mL concentration of DA wasdissolved in PAA solution, the resulting mixture became highly viscousresulting in even higher viscous solution than PAA-CA. Hence the optimumconcentration of DA needed should be less than 0.5 mg/mL concentrationin 0.20 M PAA, but 1 mg/mL works for 0.18 M PAA.

L-Alanine (A) can be used at 1 mg/mL concentration; higherconcentrations were not tested because 1 mg/mL gave desirable viscosityeven at 0.20M PAA solutions. A does not dissolve in DMAC, its insolublecrystals are visible in PAA-A solution; however it is possible some of Adissolved because in terms of mechanical properties PAA-A membrane wasgood. Its plastic-like structure did not turn brittle even at sixmonths' exposure in the hood. Similar characteristics were observed for3 weeks with PAA/PAA-W/PAA-CA composite membranes; when prepared usinglayer-by-layer casting on glass. In order to develop more durablemembranes based on FIG. 1a , PAA was incubated with GA, and PAA-W andPAA-CA were sequentially casted on PAA. The aim was to observe if GAmoves to PAA-CA solution.

Based on contact angle measurements data, this composite produced thehighest hydrophobicity among FIG. 1a membranes. This layer-by-layercasting is not similar to the technique used to prepare polyelectrolytemultilayer membranes which relies on charge-charge interactions ofdifferent layers.

L-lysine (K) shows similar pattern to A, but it makes PAA solution muchmore viscous at the same concentration. A good concentration should beless than 1 mg/mL at 0.18 M PAA. At 0.20 M PAA concentration, 0.5 mg/mLof K produced a highly viscous PAA-K solution.

L-Cysteine (C) was selected because it has a free —SH group. IntroducingC to CS membrane via GA makes CS membrane very flexible. Like-wise K, Cis not soluble in DMAC. Interestingly, it was observed that C formsfibrils in DMAC and during membrane preparation these fibrils resultedin blocks within the PAA-C membrane shown in FIGS. 62a-62g . Therefore,highly heterogeneous PAA was formed, but these membranes protect theplasticized form [FIG. 1 a i] more than the others except PAA-A [stablefor around 3 months]. However, stirring PAA-C solution at very highspeed minimized the fibril formation. It was observed that cysteinemakes wrinkled PAA membranes, however its mechanical strength was stillhigh in comparison to phase-inverted PAA membranes. When GAconcentration was 0.21% in PAA-C solution, it did not result inplasticized PAA-C membrane but it resulted in shiny and relativelystronger membranes than individual PAA membranes. So, for preparation ofPAA-C membranes, the optimum GA concentration should be between about0.21 to about 0.35%.

For the amino-acids of FIG. 66 and the other molecules utilized toprepare the disclosed PAA films, the GA concentration can be up to about2%. However, the concentration of GA depends on the formula andcomposition of the membrane.

L-isoleucine (I) can be used at less than 1 mg/mL concentration, atwhich concentration desirable PAA-I viscosity can be obtained. In thisstudy, 0.5 mg/mL-3 mg/mL were tested at 0.20M PAA solution, and in allcases the extremely high viscosity of the mixture did not allow membraneformation. However, 0.5 mg/mL I was a good concentration at 0.18 M PAAsolution for desired fluidity during membrane preparation.

Likewise, L-Tryptophane-methyl ester (W) can be used at less than 0.5mg/mL concentration, at which concentrations desirable PAA-W viscositycould be obtained. In this study, 0.2 mg/mL-1 mg/mL were used at 0.20 MPAA solution, and in all cases extremely high viscosity did not allowmembrane preparation. However, 0.5 mg/mL W could be used at 0.18 M PAAsolution to get the desired fluidity. For the same concentrations, PAA-Wgave the highest viscosity. W, gave unexpected results for FIG. 1bmembranes as well.

Glycine (G) and L-Aspartic Acid (D) exhibit similar pattern to A, butmakes PAA solution much less viscous at the same concentration, hencethe optimum concentration should be at about 1 mg/mL at 0.18 M PAA.

After PAA-W, PAA-R (L-Arginine (R)) has the second highest viscosity[these are just based on observations]. 0.2 mg/mL R can be used with0.20 and 0.23 M PAA; 0.25 M PAA. 0.5 mg R was observed to give a goodconcentration at 0.18 M PAA solution. However, the formed membranes didnot provide durable membranes; keeping the membrane at room temperaturefor 2 days made the membrane brittle.

L-Threonine (T) can be used at 0.5 mg/mL for 0.20 M PAA and 0.18 M PAAsolutions. However, similar to L-arginine, T made the PAA membranebrittle within 3 days after drying.

All these amino acids were introduced to the PAA solution immediatelyafter the formation of PAA solution as described in FIG. a. However,these biomolecules were added to the system at the same time with ODA.The resulting PAA solutions showed similar characteristics. All thesecharacterizations were made for FIG. 1a membranes; the concentrations ofthese amino acids can be increased up to 2 mg/mL in FIG. 1b . However,thiol containing amino acids and other molecules did not lead to theformation of membranes when FIG. 1b were employed.

Optimization of Cross-Linker Concentrations Optimization of GAConcentration:

For desirable PAA membranes, the following parameters were found to befactors: Concentration of GA stock [Sigma-Aldrich, 70% Glutaraldehyde]solutions, the final concentration of GA when mixed with PAA solutions,age and temperature of GA added to the PAA solutions are highlyimportant in terms of obtaining desirable PAA membranes. Age of GA is aterm used here to describe what type of pre-treatment was applied to GAbefore it was introduced to PAA solution. Temperature of GA refers tothat at which temperature GA was incubated before it was introduced toPAA solution.

There are similar procedures to age GA, but the way of aging in thisstudy is not related to time, rather it is related to temperature. AgedGA, in general, provides distinct results than fresh GA duringcrosslinking. General rules in optimization of GA can be listed asbelow: concentration of GA stock is a factor in terms of how much wateris introduced to the PAA solution; even though water provides a workingmicroenvironment to GA, it can disrupt the cross-linked PAA membraneformation and also cause localized phase inverted PAA membrane formationin the PAA solution's vial, or during the membrane formation. So, it isadvisable to use high stock concentration if the FIG. 1 a iii is used toprepare the membranes.

Concentration of GA added to the PAA solutions should not be over theconcentration at which it makes pure 0.16 M or 0.12 M PAA solutionssolid less than 15 min and 30 min, respectively. Beyond this point, theleads to the formation of easily breakable PAA membranes. Similarobservation was shown for PAA-CS.

Further optimization can be performed for each molecule accompanied withPAA.

However, it should be noted that incubation time also matters indefining the degree of cross-linking. When GA is less than 0.35%, theresulting PAA membranes are in between pure water phase inverted PAAmembrane resulting in plasticized form.

However, higher GA concentrations such as 2% GA convert PAA solutioninto completely non-fluidic within 3 min, which was observed in FIG. 1 aiii and FIGS. 1 bi and 1 bii.

Age of GA defines its active individual GA molecule and degree ofauto-polymerization. NMR characterization and further explanations areprovided above.

GA stock vial should be kept in the hood until before being placed atroom temperature for approximately 10-20 min; when the GA stock has beenleft at 4° C. needs 10 min, and it is then transitioned into roomtemperature, its viscosity decreases and becomes highly fluidic (for 25%or less forms), which was concluded based on observations throughpreparing different concentrations from 70% stock. When it is introduceddirectly from the refrigerator, it forms localized phase-inverted PAAmembranes using the PAA solution.

Another point with respect to GA is that it must be thoroughly mixedwith dry-DMAC before introducing it to PAA viscous solution. Moreover,there are two observations in this application as (i) adding GA intoDMAC containing vial causes heat formation. In that respect, DMAC shouldbe added slowly to the GA containing vial [note: the resulting heat maynot increase the temperature up to flash point of DMAC but beingcautious is advisable because mixing 25% stock GA with DMAC releasesheat causes over 40° C.]. Increase in heat is possibly related towater-DMAC interaction since the same amount of GA in less water contentadded to the DMAC released less heat. The second observation (ii) isthat using the stock solution of GA is different than GA that has beenmixed in water. Pretreating GA with DMAC gives better membranes andcauses no local membrane formation in PAA viscous solution. The PAAmembranes prepared using FIG. 1a ii were found to be more stable formonths when even kept under hood and at room temperature.Simple-treatment of GA in DMAC prior to the introduction into the PAAsolution can bring a change in the resulting membrane.

The overall result of addition of GA to PAA viscous solutions from stockGA [FIG. 1a iii] makes the PAA membranes [except PAA-Cys and PAA-A]brittle after drying. This could be related to the kinetics of membraneformation according to FIG. 1a . It should be noted that the membranesdescribed here were synthesized according to the procedure described inFIG. 1 a iii. However, the PAA membranes can be stored in pure waterwhich protects their mechanical properties or prevents them from beingbrittle. However, PAA-A and PAA-Cys membranes were found to be strongand more durable even up to months. Addition of pre-diluted GA stockwith dry DMAC to PAA solutions make PAA membranes strong and durable.

Optimization of 1, 1′-diimidazole

1, 1′-diimidazole (IZ) was employed as a solid by slowly adding thesolid particles into PAA solutions. Introducing over 1 mg/mL IZ to thePAA solutions at once causes localized orange color solid formation.However, IZ's original color is pale-yellow. Besides; IZ-treated PAAforms heterogeneous membrane, for instance the inner part looked similarto the non-cross-linker treated PAA membranes while the outer layerlooks more like a plastic [Synthesized as described in FIG. 1 a iii]. Inorder to eliminate formation of non-even membranes, IZ should be used atlow amounts and under good mixing.

IZ works slower than GA for membrane formation; while GA requires lessthan 3 min to increase the viscosity up to the desired level, IZrequires 15 min at 0.16 M PAA while 45 min requires at 0.12M PAA. Thefinal membrane is physically similar to GA-treated PAA membrane. Inorder to prepare a totally plasticized-membrane, 3 mg/mL IZ should beadded to the system under FIG. 1 a iii. Besides, imidazole was dissolvedin anhydrous DMAC and then applied to the PAA and PAA-small moleculeviscous solutions. However, there was no difference between directaddition of solid imidazole and DMAC-dissolved imidazole. Beside1,1′-diimidazole, N,N′-dicyclohexylcarbodiimide [DCC] was tested, andsimilar membranes were obtained with longer incubation times. DCCmediated membranes gave similar colored membranes as obtained with IZ.However, EDC/NHS did not work either direct solid addition, or dissolvedin water.

Likewise, EDC/NHS, the use of glutaric acid [Sigma-Aldrich, MO] did notprovide any plastic-like membranes. However, glutaric acid simplyenhanced the IZ's activity to make PAA solutions' viscosity high enoughto be prepared membrane relatively quick. It should be emphasized thatplastic-like structure does not mean just being a transparent membrane,which is common for evaporation mediated phase inversion, but that meansformation cross-linker mediated membranes. Neither carbodiimidazoles norglutaraldehyde required EDC/NHS to modify PAA molecules. Thesecombinations did not require EDC/NHS for crosslinking. Extensive testswere performed for GA, and NMR data show that GA covalently binds to PAAwithout the use of any EDC/NHS intermediate (discussed above).

Treating PAA with DCC until the PAA solution became solid resulted invibrating solid which was seen for GA treated CS solution. This effectwas not seen for GA treated PAA, and was observed for relatively lowerin the cases of IZ treated PAA. For IZ, it could be related to lower andlocalized solubility of IZ in viscous PAA solution. The vibrationproperty was not tested with an instrument: it was visually observed.

Time-Dependent Alterations in Membrane Physical Characteristics.

Incubation of PAA with a cross-linker alters its physical and chemicalcharacteristics. These changes both depend on the type of cross-linkerand the biomolecule used to modify PAA. Glutaraldehyde is highly activemolecule, and it is not easy to control its diverse binding to PAA andother small molecules. Immediately after the phase-inversion, most ofthe PAA membranes protect their flexible natures. The membranes otherthan L-cysteine, L-alanine and chitosan modified PAA membranes did notprotect their plasticized nature after about a week. However, thementioned three membranes protected their plasticized nature well over 6months. Measurement did not exceed 6 months. However, in parallel toincrease in phase inversion incubation in water they become brittle,which takes weeks to months [Synthesized as described in FIGS. 1 ai and1 aii].

Phase inversion or the process of transformation from solution state tosolid state is a technique in preparation of membranes. It is used frommicro-filtration to gas separation applications. Four main approacheshave been described for phase inversion; (i) coagulation bath mediatedphase inversion, (ii) heat triggered phase inversion, (iii)precipitation from vapor phase and (iv) evaporation in non-solvent.

Pore formation on the top layer of membrane through coagulation-bathmediated phase inversion forms due to flow of non-solvent into themembrane while the thickness of membrane's skin layer depends on flow ofsolvent from inner part of membrane into the coagulation bath. However,open-pore formation also requires coalescence in the polymer-poor areawhich is right under the top-layer. The relation between solvent andpolymer itself, and solvent and non-solvent are also important indetermining the characteristics of the pores. For example, in thepresence of high affinity solvent and non-solvent, macrovoids areformed.

FIG. 59 is an illustration of phase-inversion in coagulation-bath(immersion precipitation).

Solvent refers to the solvent used to prepare membrane solution whilenon-solvent refers to the solvent used in the coagulation bath (which isalso not supposed to be main solvent for the membrane). The solvent canbe any suitable solvent capable of dissolving other substances, such asethanol, methanol, and combinations thereof. The non-solvent can be anysuitable material that is not capable of substantially dissolving othersubstances, such as water.

Besides altering the non-solvent, addition of non-solvent into solvent(casting solution) also makes difference in pore formation. While a veryfast desolvation of solvent into non-solvent forms finger likestructures and cause no or rare pore formation, addition of solvent intonon-solvent allows porous surface formation because this situationreduces solvent desolvation into the non-solvent. However, addition ofnon-solvent into the solution before it is casted, less porous surfacewith a less dense surface forms. The solvation power of solvent alsopossesses strong effect on pore formation for instance a less solvationpower provides more pores on membrane surface.

The membrane formation process here is mostly driven by combination ofglutaraldehyde, evaporation and coagulation-bath/glutaraldehydeevaporation. When there is no glutaraldehyde in the system, even thespeed of stand-alone membrane formation through evaporation is reduced.However, it should be mentioned that this does not mean that GA enhancessolvent evaporation. Faster formation of stand-alone membrane impliesthat GA cross-links individual polymers leading into macro-polymericsystems by which stand-alone membrane formation eliminates highpercentage of DMAC removal from the system.

In the method used herein glutaraldehyde is not only functioning as across linker, it is also transforming the resulted PAA into opticallyactive form such as fluorescence active forms. These bindings alter themembrane formation; the very basic alteration is the rate of solventevaporation. Here, encrossslinking was used to refer extensivecrosslinking.

As explained for PAA-GA, PAA-CS-GA, PAA-A-GA, PAA-DA-GA and PAA-C-GA,the concentration and activity of GA (i.e. heat treatment andpre-treatment with DMAC) showed dramatic effect on the resulting PAAmembranes synthesized according to FIG. 1a , in which coagulation bathwas used as the final step of the phase-inversion.

For some small molecules, the membrane showed thin inner solid layerwhich was then disappeared when the membrane was incubated under hoodfor 6 h or more, which refers to a continuing progress of membraneformation. However, in the cases of higher GA concentrations and/orenhanced activity of GA, formation of plastic-like and transparentmembrane surface didn't show any requirement to sonication inmethanol/ethanol or drying under hood. It is evident that GA alters thekinetics of membrane formation; additives were shown a role-player inthe kinetics. In the cases of the membranes synthesized according toFIG. 1b , all of the membranes are plastic-like and transparent, anddon't form any solid amorphous layer even they are submerged into wateror other solvents.

In FIG. 1 a membranes, there is no pore formation in the cases of waterbath becomes the final phase-inversion step. Therefore, the speed ofDMAC releases into water bath is too fast to form porous surface, butthe surface becomes shiny and plastic-like which can be assigned to theactivity of GA. However, when the final step of phase-inversion wasperformed in methanol, the surface lost its shiny structure and formedwoven-like structures (as discussed above) which can be explainedthrough DMAC having a relatively lower tendency to diffuse intomethanol.

Similarly, losing the shiny outer surface appearance can be explained bythe quenching activity of methanol on the activity of GA crosslinking.However, when methanol/ethanol bath was accompanied with sonication,membranes then can be synthesized porous. In the cases of high GAconcentrations, the membranes can also be made porous if they aresonicated in methanol/ethanol bath. In essence, surface patterns of FIG.1a membranes depend on the final step of phase-inversion while thetexture of them is mostly dependent on concentration and treatment ofGA. However, it should be noted that small molecule also makesdifference in formation of the membrane such as PAA-C-GA, PAA-W-GA andPAA-A-GA are more of on the plastic-like surface forming co-polymerswhile chitosan and glucosamine urge the membranes become more ofamorphous.

Another test was performed to determine how glutaraldehyde, smallmolecule and the final step of phase-inversion affect the final textureof PAA membranes. The co-polymers were left in a vial overnight and theresulted PAA co-polymers were sticky gels. Then, they were exposed toevaporation under hood, phase-inversion in water-bath, methanol,methanol-water mixture, and ethanol and ethanol-water mixtures. PAA-W-GAand any other PAA co-polymers enhanced with sulfanilic acid providedplastic-like membrane surfaces under all final phase inversionconditions, but only sonication in organic solvent (i.e. methanol andethanol) made the membranes fully plastic-like; as shown for FIG. 1bmembranes, high amount of residual DMAC in PAA makes it possessing solidamorphous layer.

This is evidence that the speed of solvent removal from the inner partof the membrane is one of the parameters in order to form totallyplastic-like membranes. Besides, sonication allowed the membranespossess porous surfaces in organic solvents; even though ethanol worksbetter such as 80% ethanol is enough for porous surfaces while methanolcan be 100%, methanol bath treated membranes showed better durability.Besides, introduction of organic solvents such as ethanol, hexane andothers into co-polymerization media did not affect the pore formation aswell. Introducing solvent into coagulation bath was shown a parameter tomake flat membranes porous, but the approach did not work for themembranes synthesized according to FIG. 1 a.

Classical pore preserving agents include PEG 400 or any other agentsweren't tested to make the membranes porous since the goal was just tounderstand how membrane formation progresses under different conditions.

Preparation of Ternary PAA Membranes

Images of several films are shown in FIGS. 60a-60h . FIG. 60a :PAA-CS-GA; FIG. 60b : PAA-A-GA; FIG. 60c : PAA-A-GA; FIG. 60d :PAA-A-GA; FIG. 60e : PAA-GA; FIG. 60f : PAA-A-GA; FIG. 60g : PAA-GA andFIG. 60h : PAA-CS-GA. All of these digital images were taken between30-60 min right-after castings on the glasses. Steps of thephase-inversion according to FIG. 1a resulted in differentsurface-properties.

The casted solutions shown in FIGS. 60a-60h underwent different finalphase-inversion process, by which different surface properties of sametypes of membranes were obtained. FIGS. 60i-60p illustrates-PAA-CS-GAFIG. 60a was incubated for 6 h at room temperature, followed byincubated at 70° C. for 20 min. The resulted membrane turned intobrownish colored transparent and brittle membrane FIG. 60i . PAA-A-GAFIG. 60a was incubated at room temperature for overnight FIG. 60j .PAA-A-GA FIG. 60c was incubated at room temperature for 6h, followed byincubated at 70° C. for 1h.

The resulted membranes showed quite-similar characteristics withPAA-CS-GA membrane FIG. 60i . PAA-A-GA FIG. 60d was incubated atroom-temperature for 3h, followed by incubated in 100% methanol for 1 h.The resulted membrane FIG. 60l showed opaque and non-shiny surface withpossessing plastic-like edges. PAA-CS-GA FIG. 60h was incubatedovernight at room temperature, and the resulted membrane possessedslightly shinny surface FIG. 60m . Since the thickness was over 0.3 mm,total transparency was not obtained.

PAA-GA FIG. 60e was incubated at room temperature for 6h, and theresulted PAA-GA FIG. 60n showed shinny surface. PAA-A-GA was incubatedat room temperature for overnight, and the resulted membrane FIG. 60oshowed slightly shiny color. PAA-GA FIG. 60g was incubated at roomtemperature overnight at room-temperature, and the resulted membraneFIG. 60p showed shiny surface. This membrane did not show anyplastic-like surface how it was seen for the membrane FIG. 60n , whichcan be attributed that the edges of FIG. 60n was thinner while thecenter was thicker. However, for the PAA-GA membrane FIG. 60p , thesolution was evenly distributed. All of these membranes were preparedaccording to FIG. 1a , which means the last step of phase-inversion tookplace in pure water if not specified otherwise.

FIGS. 61a-61e includes several images. FIG. 61a PAA-A-GA (0.25%); FIG.61b PAA-A-GA (0.75%); FIG. 61c PAA-C-GA (0.25%); FIG. 61d PAA-CS-GA(0.3%), FIG. 61e PAA-GA (0.25%). 70% stock GA was pre-dissolved in DMACif not mentioned otherwise. All of the membranes prepared according toFIG. 1 a.

FIGS. 62a-62g includes several images. FIG. 62a —PAA-GA; FIG. 62b—PAA-DA-GA; FIG. 62c —PAA-A-GA; FIG. 62d : PAA-DA-GA (direct from 70%stock); FIG. 62e —PAA-A-GA was first incubated in 70° C. for 30 min,followed by overnight incubation at room temperature; FIG. 62f —PAA-A-GAsimilar to c but higher GA concentration (% 0.9); FIG. 62g —PAA-C-GA.70% stock GA was pre-diluted in DMAC if not mentioned otherwise,followed by introduced to the membrane formation processes at theconcentration of 0.3% if not mentioned otherwise.

GA concentration and its form are important in terms of membranecharacteristics. Besides, viscosity of PAA solution is important. Asseen in FIG. 60g , PAA-DA gave some blue region but the rest isyellowish. Interestingly, increased incubation time and high GAconcentration make the membranes plastic like-structures and colorful.However, this is not clear during phase-inversion. When they are gettingdry, their transparent natures become visible. At low GA concentrationand short-incubation time, the membranes don't turn into transparentform, but a thin layer forms on top of the membranes. The bottom of themembrane is mostly not-shinny. It should be mentioned that the way of GAapplication is important in membranes' mechanical and opticalproperties. For example, while PAA-CS forms strong-green color membranewith FIG. 1 aii, it does form pale chestnut color with FIG. 1 ai. Forexample, FIG. 13a illustrates UV/Visible spectroscopy for severaldisclosed films. The Y-axis in the data is referred to as OpticalDensity, or DO, with a comparatively good number between 0.1 and 0.9.The larger the number the stronger the color intensity.

FIGS. 63a-63k are images of the following films: FIG. 63a : PAA-pAS-GA;FIG. 63b :PAA; FIG. 63c : pAB-PAA-GA; FIG. 63d : pAB-DMAC-GA-PAA; FIG.63e : W-GA (long incubation)-PAA: FIG. 63f : W-GA-PAA; FIG. 63g :pAB-DMAC-GA-PAA (30 min incubation); FIG. 63h : PAA-pAS-W-GA; FIG. 63j :pAS-DMAC-GA-PAA; FIG. 63k : PAA-pAB-GA. All of the membranes preparedaccording to FIG. 1 b.

0.32 M highly viscous (no fluidity) was treated with 0.25% GA for 6 h atroom temperature, followed by casted on glass and phase-invertedaccording to FIG. 1a . The resulting films are shown in FIG. 64a finalsteps of phase inversion were in 100% Ethanol, FIG. 64b 50%Ethanol:water and (FIGS. 64 c/d) and nano-pure water. All of themembranes prepared according to FIG. 1 a.

PAA-GA

As seen from FIG. 60e and FIG. 60g , GA (aged) treated PAA showedsimilar transparent-view while they ended up showing distinct surfacesin response to alteration in the final step of phase-inversion (FIGS.60n and 60p respectively). Both of the membranes were synthesizedaccording to FIG. 1a iii, but completion of the phase inversion wasperformed in methanol (FIGS. 60 g/p) in addition to water-bath (membraneshown in FIGs. e/n). Thinner regions of the membrane FIG. 60n showedfully-plastic-like structure while the thicker regions showed twodifferent characters; the outer part was fully plasticized while theinner part was amorphous. However, the membrane shown in FIG. 60p wasmore of amorphous membrane and no plastic-like outer layer was seen.Contact angles of the membranes showed difference as well; they were 67and 61 for the membranes shown in FIGS. 60n and 60p , respectively.However, when PAA-GA showed total plastic-like such as the edges of themembrane FIG. 60n was 58. When the GA was diluted in DMAC before it wasapplied to PAA solution brought strong impact on membrane formation(shown in FIG. 60a ). GA from 70% stock was pre-dissolved in DMAC,followed by introduced to the PAA solution. The membrane was preparedaccording to FIG. 1a ii with 0.9% GA. The resulted PAA gave durable andfully plastic-like membrane. Even though, the PAA-GA (0.25%) membraneshown in FIG. 61e was prepared according to FIG. 1a ii, the resultedmembrane showed totally different character; outer layer and thinnerregions were plastic like while the center was amorphous because of thethickness.

PAA-CS-GA:

Here three different PAA-CS-GA were shown how glutaraldehyde affectedcolor formation and formation of plastic-like structure. As seen fromFIGS. 60a and 60h , both PAA-CS-GA were transparent. (FIG. 60a )PAA-CS-GA (% 0.25) was first incubated under hood for 6h, followed byincubated at room temperature for overnight (˜14h). However, (FIG. 60h )PAA-CS-GA (% 0.5) was only incubated at room temperature for overnight(˜20h). Phase-inversion of both membranes was finalized in water-bath,followed by dried under hood for 2h. As seen from FIG. 60i , themembrane (FIG. 60a ) resulted in a brownish plastic-like membrane whilethe membrane (FIG. 60h ) resulted in greenish amorphous membrane (FIG.60m ). For both of these membranes, aged-GA was directly introduced tothe membrane formation process. However, GA from 70% stock waspre-diluted in DMAC, followed by warmed for 10 min at 70° C., which wasthen introduced to PAA-CS solution seen in FIG. 61d . The membrane (FIG.61d ) was incubated at room temperature for 15 h, and phase inversionwas completed in water-bath for 2 h followed by dried under hood for 2h.The resulted PAA-CS-GA membrane (FIG. 61d ) was durable, strong andresistant to the organic solvents while the membrane (FIG. 61m ) becamebrittle within three days. The membrane (FIG. 61m ) did not turn intoplastic-like membrane within 2 weeks, but rather the membrane becamebrittle. The membrane (FIG. 61d ) did not form a transparent greenishmembrane at once, rather it took over 2 h to form the transparentmembrane; at first it was opaque and within time it turned intotransparent form.

PAA-A-GA

FIGS. 60b, 60c, 60d and 60f are showing the membranes before theyunderwent final step of the phase-inversion. Even though same GA (stock25%) was used for these four membranes, heat treatment and alteration infinal step of phase-inversion showed strong impact on physicalproperties of final membrane. Heat treatment at 70° C. for 1 h wasapplied to the casted PAA-A-GA solution before it underwent incubationat room temperature. The resulted membrane showed fully plastic-likereddish structure (FIG. 60k ), but it was not durable and became brittlewithin a week. When the completion of phase-inversion took place inmethanol/water mixture, the resulted PAA-A-GA (FIG. 61) was opaque. Incontrast to this, outer layer of the PAA-A-GA (FIG. 60k ) wasplastic-like and shiny. However, when the membrane was thick, the outerlayer did not provide similar plastic-like view, rather it gave a shinysurface (FIG. 60o ). When 70% GA was dissolved in DMAC, followed byintroduction to PAA-A solution, it affected membrane formation; beforethe membrane formation underwent incubation at room temperature, it waswarmed at 70° C. for 10 min. The resulted membrane (FIG. 61a ) showedthicker plastic-like outer layer in comparison to the membrane shown inFIG. 61b . Similarly, low GA concentration and short incubation (6htotal) resulted in non-plastic like membrane formation (FIG. 62c ). Theedges of PAA-A-GA (FIG. 61a ) are totally transparent, and the overalldurability of the membrane is better than FIG. 60j membrane. When GAconcentration was increased (0.75%), the resulted PAA-A-GA becametransparent and brownish (FIG. 62b ); the membrane was durable andstrong. Similarly, when the GA concentration was increased from 0.75% to0.9%, the resulted membrane became yellowish (FIG. 62f ) and bettertransparency in comparison to the PAA-A-GA membrane shown in FIG. 61b .Heat treatment allowed PAA-A-GA (FIG. 62e ) totally shiny andplastic-like view, which was also durable in comparison to heattreated-PAA-GA (has similar color).

PAA-C-GA and PAA-DA-GA

Dilution of GA in DMAC totally altered the view of PAA-DA-GA even thoughsame GA concentration was used. PAA-DA-GA (FIG. 62d ) showedplastic-like outer layer and stronger and durable membrane. However, asshown in FIG. 60m , fully plastic-like membrane can be obtained ifPAA-DA-GA was heated for 20 min as of first step incubation at 70° C.Heat treatment allowed PAA-C-GA (FIG. 60i ) membrane became totallyplastic-like structure, but not very transparent. When there is no heattreatment and high GA level, PAA-C-GA (FIG. 60m ) did not end up formingtransparent membrane. Glucosamine might react with GA first over C1-OHin addition to amino group, followed by introduced to PAA through aminogroups on PAA. This could be a type of Maillard reaction, which givesbrown-color formation (FIG. 62d ).

PAA-pAB-GA, PAA-W-GA, PAA-pAS-GA

pAB is also another small molecule altered overall view of PAA. In allcases, aged GA was used for preparation of PAA-pAB-GA membranes. As seenfrom the FIG. 63c , PAA-pAB-GA is bluish with 0.25% GA concentrationwhile the PAA-pAB-GA (FIG. 63k ) is more of brownish with 0.5% GA, whichunexpectedly became brittle within a month. Similar to high GAconcentration, incubation of pAB with GA in DMAC before they wereintroduced to PAA solution resulted in color changes of PAA-pAB-GAmembranes; pAB was pre-treated with GA in DMAC for 10 min and 20 min forthe membranes seen in FIGS. 63d and 63g . Similarly, W was pre-treatedwith GA for 20 min and 30 min before they were added to PAA solutionsresulted in yellowish (FIG. 63f ) and reddish membrane formation (FIG.63e ), respectively. pAS was pretreated with GA in DMAC, followed byadded to PAA solution, gave yellowish view (FIG. 63j ) while PAA-pAS-GA(FIG. 63a ) gave light purple view. However, when GA treated pAS and GAtreated W were simultaneously added to PAA solution, the membrane gavelight yellowish view (FIG. 63h ) which resembles to PAA membranephase-inverted under hood (FIG. 63b ). All of these membranes weresynthesized according to FIG. 1 b.

FIGS. 65a-65d are images of: FIG. 65a PAA (ODA+PMDA)-GA-SA 1 hincubation at room temperature, followed by phase-inversion in purewater; FIG. 65b PAA (PDA+PMDA)-GA-SA 1 h incubation at room temperature,followed by phase-inversion in pure water; FIG. 65c PAA (ODA+PMDA)-GA-W1h incubation at room temperature, followed by phase-inversion in purewater; FIG. 65d PAA (PDA+PMDA)-GA-SA 30 min incubation at roomtemperature, followed by phase-inversion in pure water. In order to gettransparent membranes, in the cases of PDA as amine sources less timerequired due to the fact that PDA has more reactive amino groups thanODA because of conjugation. Small molecule such as W resulted in bluishnon-transparent while SA provided highly transparent membrane; PAA-SA-GAgave glassy brittle membrane while PAA-W-GA gave amorphous durablemembrane.

1.13 Application of PAA Membranes for Food Packaging

Cheese, pepperoni, apple and walnut were used to test packagingproperties of the membranes.

The formed films are shown in FIGS. 67a-67e , with FIG. 67a POLLY-Opart-skim mozzarella cheese (Campbell, N.Y.); FIG. 67b Merve pepperoni(NJ); FIG. 67c Cabot extra sharp cheddar cheese (Cabot, Vt.); FIG. 67dGreen apple (WalMart, Jonson City N.Y.) and FIG. 67e Diamond walnut(CA). PAA-A-GA, PAA-A-pAS and PAA-I-pAS membranes were used forpackaging, respectively.

The films were sterilized rinsing 70% Ethanol, followed by rinsing withexcess pure water (18.2 MΩ), which were finally treated with 1 h UVlight. Food samples were kept in fridge at 4° C. Cheeses and pepperoniprotected their stability for the tested period, 3-6 months.

FIGS. 68a-68c are images of the stored foods of FIGS. 67a-67c . FIGS.68a and 68c are images of cheeses stored for 10-11 months. As it isseen, the microbial growth is localized in FIG. 68a , which wasintentionally pierced.

FIG. 68c is an image of pepperoni after 15 months incubation. Nomicrobial growth was observed.

FIG. 69 illustrates the disclosed film concept for both detection andpackaging. Step 1: air packed or vacuum packed food sample producesvolatile organic compounds or other compounds; (ii) the VOC interactswith PAA, (ii) this results in color-change that is visually detected orelectronically detected.

The disclosed films can be used as a packaging material but at least aportion of the film makes no direct contact with the food sample. Anyvolatile or semivolatile organic vapor that is produced as a result offood spillage is drawn on to the sensor/packaging PAA. The sensorresponds via a visible color change and a measurable change inconductivity using an optional conductivity monitor that is placed onthe package. The concentration of the emission of volatile organiccompounds (VOCs—e.g., sulfur compounds, acetone, methyl ethyl ketone,toluene, ethylbenzene, m,p-xylene, styrene, and o-xylene) largelyincreased over the storage time and should be correlated with the totalnumber of microbial numbers. This should allow a rapid detection of foodspoilage and may also allow consumers to visually determine foodfreshness. The PAA film can also detect pH-related changes in the airaround the food (e.g. ammonia, alcohol).

1.14 Thermoplastic Examples

This example includes formulations that are capable of forming a PAAfilm that i) softens when heated (thus allowing the film to be molded todifferent shapes and sizes); ii) is flexible and undergoescrystallization transitions by incorporating sulfur-containing monomers,fatty acids, ionic salts and liquids, and plasticizers between thedifferent functional groups; and iii) is resistant to shrinking whileretaining good strength and chemical stability.

In this example, these films can exclude, wholly or partially, theformation of covalent bonds while increasing ionic properties,mechanical strength and dissolution. The resultant film in this exampleis referred to as “Thermoplastic PAA”.

Unlike a “thermoset” PAA polymer that is held together via irreversiblechemical bonds, Thermoplastic PAA is relatively weakly held togetherthrough electrostatic interactions and Van der Walls forces. Theserelatively weak bonds in the thermoplastic polymers allow them to bere-usable, relatively soft when heated, and to be molded and remoldedone or more times.

This ability to reuse thermoplastic typically means a higherrecyclability. Also, other properties such as good strength and atendency to resist shrinking is realized by these Thermoplastic PAAfilms.

These Thermoplastic PAA films can be made more thermoplastic by reactingsulfur containing monomers (e.g. 4,4′-thiodianiline; an analogue of4,4′-oxydianiline) in a stoichiometric ratio of acid/amine functionalityand other additives. Examples of Thermoplastic PAA Films include but arenot limited to those shown in Table S.

TABLE S Examples of Proposed Thermoplastie PAA Film Formulations

adipate (DMAD) to PAA

Various Thermoplastic PAA films can be developed using variouscalculated concentrations of acid/amine, plasticizers, and monomers.These concentrations can be derived using the concept of criticalbranching coefficient. Mixtures of Thermoplastic PAA films canincorporate plasticizers (e.g. adipates, phthalates, and citrates)and/or two polymer chains (e.g PAA and chitosan) interacting viahydrogen bonding and electrostatic forces. The main polymer chains canmove freely using these formulations.

Additional formulations can include the use of shorter or longer alkylchains and a range of other dicarboxylic acids (e.g. oleic acids,palmitoleic acid, sapienic acid, and linoleic acid). Other materials tobe added for the formation of the Thermoplastic PAA films can includelow to high polarity esters (e.g. nitriles, polychloroprene, chlorinatedpolyethylene and epichlorohydrins) in order to decrease the attractionbetween polymer chains to make them more flexible. A range of esters(e.g. sabacates, terephthalates, gluterates and azelates) are options.These polymers can be synthesized in environmentally-friendly solvents.

The Thermoplastic PAA films can be analyzed in several ways, for exampletheir structures can be characterized using 1H and 13C Nuclear MagneticResonance (1H NMR) Spectroscopy and Heteronuclear Single QuantumCoherence (HSQC) spectroscopy of the 1H-13C system. The polymerizationcan be validated via Infrared Spectroscopy (IR) by analyzing changes inthe functional groups. The molecular weights can be determined via sizeexclusion chromatography. Also, Differential Scanning Calorimetry (DSC)can be used to study the thermal transitions of the polymers.

These Thermoplastic PAA films exhibit a decrease in the glass transitiontemperature (Tg) for the films containing plasticizers in their DSCcurves. For all formulations, the appearance of a crystallizationtemperature (Tc) peak is evident in the DSC curves. This peak indicatesthe crystallinity of the polymers upon cooling, which also suggeststhermoplasticity. Also, these films have an increase in plasticity and areduction in rigidity due to an absence or a relatively low amount ofcovalent crosslinking.

Throughout the application the following acronyms are used whendiscussing PAA films. The meaning of these abbreviations appears below:

PAA: Poly(amic) acid

GA: Glutaraldehyde A-alanine W-tryptophane CS-Chitosan

SA-Sulfanilic acid

I-isoleucine K-L-Lysine

CA-cellulose acetatepAS-p-aminoscalicylic acidPDA-PAA-p-phenylenedianiline+pyromellitic dianhydride PAA

IZ-Carbodiimizole

pAB-: p-aminobenzoic acidPCL-3-chloro-4-aminobenzoic acid

C-cysteine

BB-2 benzoylbenzoic acid5AS-5-aminosalycylic acid4AS-p-aminoscalicylic acid

Ser-L-Serin DA-D-glucosamine SN-sulfanilamide T-L-Threonine

The described embodiments and examples of the present disclosure areintended to be illustrative rather than restrictive, and are notintended to represent every embodiment or example of the presentdisclosure. While the fundamental novel features of the disclosure asapplied to various specific embodiments thereof have been shown,described and pointed out, it will also be understood that variousomissions, substitutions and changes in the form and details of thedevices illustrated and in their operation, may be made by those skilledin the art without departing from the spirit of the disclosure. Forexample, it is expressly intended that all combinations of thoseelements and/or method steps which perform substantially the samefunction in substantially the same way to achieve the same results arewithin the scope of the disclosure. Moreover, it should be recognizedthat structures and/or elements and/or method steps shown and/ordescribed in connection with any disclosed form or embodiment of thedisclosure may be incorporated in any other disclosed or described orsuggested form or embodiment as a general matter of design choice.Further, various modifications and variations can be made withoutdeparting from the spirit or scope of the disclosure as set forth in thefollowing claims both literally and in equivalents recognized in law.

1.-37. (canceled)
 38. A film comprising: polyamic acid (PAA);glutaraldehyde (GA); and one or more molecule selected from the groupconsisting of alanine (A), tryptophan (W), 2-benzoylbenzoyl (BB),Polycaprolactone (PCl), L-Cysteine (C), D-glucosamine (DA), dipropyleneglycol (DP), p-aminobenzoic acid (pAB), L-isoleucine (I),p-aminosalicylic acid (pAS), sulfanilic acid (SA), and 5-aminosalicylicacid (5AS).
 39. The film of claim 38, wherein the one or more moleculeis selected from the group consisting of sulfanilic acid (SA),p-aminosalicylic acid (pAS) and 5-aminosalicylic acid (5AS).
 40. Thefilm of claim 38, wherein the one or more molecule is selected from thegroup consisting of alanine (A), tryptophan (W), 2-benzylbenzoyl (BB),L-Cysteine (C), D-glucosamine (DA), dipropylene glycol (DP),p-aminobenzoic acid (pAB), L-isoleucine (I), p-aminosalicylic acid(pAS), and 5-aminosalicylic acid (5AS).
 41. The film of claim 38,wherein the film has a thickness of about 0.02 mm to about 0.12 mm. 42.The film of claim 38, wherein the film has a modulus of elasticity ofabout 2.2 GPa to about 4.1 GPa.
 43. The film of claim 38, wherein thefilm has a tensile strength of about 59.9 MPa to about 95.1 MPa.
 44. Thefilm of claim 38, wherein the film has an antibacterial activity ofreducing the number of colony forming units (CFUs) by up to about 90%.45. The film of claim 44, wherein the antibacterial activity against oneor more of gram-positive and gram-negative bacterial species.
 46. Thefilm of claim 45, wherein the gram-positive species is Staphylococcusepidermidis and Listeria monocytogenes.
 47. The film of claim 45,wherein the gram-negative species is one or more of Escherichia coli,Enterobacter aerogenes, Aeromonas hydrophila, and Citrobacter freundii.48. The film of claim 38, wherein the film is synthesized from a solventand does not include any petrochemical material.
 49. The film of claim38, wherein the solvent is ethanol.
 50. The film of claim 38, whereinthe film further comprises water.
 51. The film of claim 38, wherein thefilm further comprises acetic acid.
 52. The film of claim 38, whereinthe concentration of GA is between about 0.21% and about 0.35%.
 53. Thefilm of claim 38, further comprising one or more of an adipate, aphthalate, a citrate and chitosan.
 54. The film of claim 38, furthercomprising one or more of an oleic acid, a palmitoleic acid, a sapienicacid, a linoleic acid, a nitrile, a polychloroprene, a chlorinatedpolyethylene, an epichlorohydrin, a sabacate, a terephthalate, agluterate, and an azelate.
 55. A film comprising: polyamic acid (PAA); across-linker; and a small molecule, wherein the small molecule is amolecule having a molecular weight of less than about 900 Daltons. 56.The film of claim 55, wherein the cross-linker comprises one or more ofglutaraldehyde and carbodiimidazole.
 57. The film of claim 55, whereinthe small molecule is one or more molecules selected from the groupconsisting of sulfanilic acid (SA), p-aminosalicylic acid (pAS) and5-aminosalicylic acid (5AS).
 58. The film of claim 55, wherein the smallmolecule is one or more molecules selected from the group consisting ofalanine (A), tryptophan (W), 2-benzylbenzoyl (BB), L-Cysteine (C),D-glucosamine (DA), dipropylene glycol (DP), p-aminobenzoic acid (pAB),L-isoleucine (I), p-aminosalicylic acid (pAS), 5-aminosalicylic acid(5AS).
 59. The film of claim 55, wherein the small molecule is one ormore molecules selected from the group consisting of alanine (A),tryptophan (W), 2-benzylbenzoyl (BB), Polycaprolactone(PCl), L-Cysteine(C), D-glucosamine (DA), dipropylene glycol (DP), p-aminobenzoic acid(pAB), L-isoleucine (I), p-aminosalicylic acid (pAS), sulfanilic acid(SA), 5-aminosalicylic acid (5AS).
 60. The film of claim 55, wherein thefilm further comprises water.
 61. The film of claim 55, wherein the filmfurther comprises acetic acid.